Anti-CD98 antibody

ABSTRACT

A human antibody or a functional fragment thereof having specific binding ability to CD98 which is derived from the cell membrane of cancer cells and is in the form of a complex with a protein having an amino acid transporter activity (for example, LAT1) is disclosed. This antibody binds to CD98 in the form of a dimer with LAT1 on the surface of cancer cells, specifically attacks cancer cells expressing CD98 via the immune system by ADCC or CDC, and further inhibits amino acid uptake of the cancer cells via LAT1, to suppress growth of the cancer cells. Accordingly, a preventive and therapeutic agent for cancer comprising this antibody or a fragment thereof, which acts on various cancers, is specific to cancer, and causes no side effect, is provided.

REFERENCE TO RELATED APPLICATION

The present patent application claims the priority of Japanese PatentApplication No. 2006-105013 (filing date: Apr. 6, 2006). The entirecontent disclosed in Japanese Patent Application No. 2006-105013 isincorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to the results of the development involvedin commission of new technology development concerning “anti-amino acidtransporter protein antibody anticancer drug” assigned by Japan Scienceand Technology Agency.

FIELD OF THE INVENTION

The present invention relates to a monoclonal antibody having a specificbinding ability to CD98 which is derived from the cell membrane ofcancer cells and is in the form of a complex with a protein having anamino acid transporter activity, and pharmaceutical use thereof forsuppression of tumor growth or cancer therapy.

BACKGROUND ART

Cancer (malignant tumor) is the primary cause of death in Japan. Thenumber of cancer patients has been increasing year by year, and thereare strong needs for development of drugs and therapeutic methods havinghigh efficacy and safety. Conventional anticancer agents frequently havelow ability to specifically kill cancer cells and act even on normalcells, leading to a great number of adverse drug reactions. Recently,development of anticancer agents targeting a molecule that is highlyexpressed in cancer cells (cancer-related antigen) has been progressedand these drugs have become effective therapeutic agents for leukemia,breast cancer, lung cancer, and the like.

It has been shown that an antibody that specifically binds to acancer-related antigen expressed on the cell membrane attacks cancercells via immunoreaction of antibody-dependent cellular cytotoxicity(ADCC), complement-dependent cell-mediated cytotoxicity (CDC) or thelike, or suppresses cell growth signaling required for growth of cancercells, and thus is useful for cancer therapy.

However, antibodies are used only for the treatment of limited types ofcancers such as breast cancer, refractory chronic lymphoma, non-Hodgkinlymphoma, acute myelogenous leukemia and the like, and there is still noantibody that can be used alone for the treatment of various types ofcancers. Accordingly, there is a demand to obtain an antibody that bindsstrongly to various types of cancer cells and has an anti-canceractivity.

CD98 (4F2) is a type II transmembrane glycoprotein chain of about 80 kDacomposed of 529 amino acid residues, which is known to be highlyexpressed in various types of cancer cells. CD98 forms a heterodimerwith a protein of about 40 kDa having an amino acid transporter activityvia a disulfide bond and is expressed on the cell membrane. Six types ofamino acid transporter proteins that are considered to bind to CD98 areknown. Although CD98 is identified as a lymphocyte activation antigen,it is considered to be involved in a great number of biologicalfunctions such as cell growth signaling, integrin activation, cellfusion and the like (Haynes B. F. et al., J. Immunol., (1981), 126,1409-1414, Lindsten T. et al., Mol. Cell Biol., (1988), 8, 3820-3826,Teixeira S. et al., Eur. J. Biochem., (1991), 202, 819-826, L. A. DiazJr. et al., J Biol Regul Homeost Agents, (1998) 12, 25-32).

Cancer cells have various mechanisms to ensure its dominance in thegrowth. For example, cancer cells overexpress neutral amino acidtransporter in order to preferentially uptake essential amino acidsnecessary for the growth over surrounding cells; which is considered tobe one of such mechanisms. L-type amino acid transporter 1 (LAT1), anamino acid transporter that is specifically and highly expressed incancer cells, was recently cloned (Kanai et al., J. Biol. Chem. (1998),273, 23629-23632). LAT1 forms a complex with CD98 and transports neutralamino acids having large side chains, such as leucine, valine,phenylalanine, tyrosine, tryptophan, methionine, histidine and the likein a sodium ion-independent manner. In addition, it is known that LAT1is poorly or not expressed in most normal tissues except for the brain,placenta, bone marrow and testis, but its expression increases togetherwith CD98 in tissues of human malignant tumors such as colorectalcancer, gastric cancer, breast cancer, pancreatic cancer, renal cancer,laryngeal cancer, esophageal cancer, lung cancer and the like (Yanagidaet al., Biochem. Biophys. Acta, (2001), 1514, 291-302). It has beenreported that when expression of LAT1 is reduced to suppress amino aciduptake, growth of a tumor is suppressed in a mouse model transplantedwith cancer (Japanese Patent Laid-Open No. 2000-157286), and suppressionof LAT1 activity is thus considered to be promising for cancer therapy.

With respect to antibodies against human CD98, a mouse monoclonalantibody that is prepared by immunizing a non-human mammal such as amouse with a human CD98-expressing cell line has been reported (Hayneset al (ibid.), Masuko T. et al., Cancer Res., (1986), 46, 1478-1484, andFreidman A W. et al., Cell. Immunol., (1994), 154, 253-263). It has notbeen known, however, whether or not these anti-CD98 antibodies suppressamino acid uptake by LAT1. Further, although an antibody against theintracellular region of LAT-1 has been obtained, no antibody that canbind to LAT1 present on the cell membrane of a living cell has beenreported. Accordingly, if an antibody that can bind to CD98 or LAT1expressed on the cancer cell membrane to suppress amino acid uptake byLAT1 is obtained, the antibody is considered to be an excellent cancertherapeutic agent against cancers in a broad range.

SUMMARY OF THE INVENTION

The present inventors have now successfully obtained an antibody havingspecific binding ability to CD98 which is derived from a cell membraneof a cancer cell and is in the form of a complex with a protein havingan amino acid transporter activity, and found that the antibody has aneffect of suppressing the growth of cancer cells, and thus is useful asan active ingredient of a pharmaceutical composition, more specificallyas an active ingredient of a preventive or therapeutic agent for tumors.The present invention is based on such findings.

Accordingly, an object of the present invention is to provide a humanantibody having specific binding ability to CD98 which is derived from acell membrane of a cancer cell and is in the form of a complex with aprotein having an amino acid transporter activity, and a functionalfragment thereof.

Another object of the present invention is to provide a pharmaceuticalcomposition or a preventive or therapeutic agent for tumors, comprisingthe human antibody and a functional fragment thereof according to thepresent invention as an active ingredient.

The human antibody and a functional fragment thereof according to thepresent invention is characterized by having specific binding ability toCD98 which is derived from a cell membrane of a cancer cell and is inthe form of a complex with a protein having an amino acid transporteractivity.

Further, the pharmaceutical composition or the preventive or therapeuticagent for tumors according to the present invention comprises the humanantibody and a functional fragment thereof according to the presentinvention as an active ingredient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the binding of human anti-CD98 monoclonal antibodies to ahuman CD98/human LAT1-expressing CT26 cell line.

FIG. 2A shows the binding of human anti-CD98 monoclonal antibodies to ahuman CD98-expressing L929 cell line.

FIG. 2B shows the binding of human anti-CD98 monoclonal antibodies to ahuman CD98-expressing L929 cell line.

FIG. 3 shows the binding of human anti-CD98 monoclonal antibodies to atunicamycin-treated K562 human cell line.

FIG. 4A shows the binding of human anti-CD98 monoclonal antibodies tovarious mouse/human chimera CD98-expressing L929 cell lines.

FIG. 4B shows the binding of human anti-CD98 monoclonal antibodies tovarious mouse/human chimera CD98-expressing L929 cell lines.

FIG. 5 shows activities of human anti-CD98 monoclonal antibodies tosuppress the amino acid uptake by a T24 human bladder cancer cell line.

FIG. 6A shows the binding of human anti-CD98 monoclonal antibodies tohuman peripheral blood T cells, B cells, and monocytes.

FIG. 6B shows the binding of human anti-CD98 monoclonal antibodies tohuman peripheral blood T cells, B cells, and monocytes.

FIG. 7 shows the binding of human anti-CD98 monoclonal antibodies toPHA-activated human peripheral blood T cells and B cells.

FIG. 8 shows the binding of human anti-CD98 monoclonal antibodies tohuman aortic endothelial cells (HAEC) and a human colorectal cancer cellline (DLD-1).

FIG. 9A shows the binding of human anti-CD98 monoclonal antibodies tovarious cancer cell lines.

FIG. 9B shows the binding of human anti-CD98 monoclonal antibodies tovarious cancer cell lines.

FIG. 10A shows the binding of human anti-CD98 monoclonal antibodies tovarious cancer cell lines.

FIG. 10B shows the binding of human anti-CD98 monoclonal antibodies tovarious cancer cell lines.

FIG. 11 shows the tumor volume in each of nude mice observed when humananti-CD98 monoclonal antibody K3, 3-69-6, or C2IgG1 was administered tothe mice to which tumor cells had been transplanted.

FIG. 12 shows the result of measurement of tumor volume afteradministration of a human anti-CD98 monoclonal antibody C2IgG1 at 100μg/mouse 3 times every other day to cancer-bearing mice having a tumorgrown to 90 mm³.

FIG. 13 shows the cross-reaction of human anti-CD98 monoclonalantibodies K3 and C2IgG1 with a monkey cell line COS-7.

FIG. 14 shows the result of measurement of tumor size afteradministration of a human anti-CD98 monoclonal antibody C2IgG1 andRituximab at 100 mg/mouse 3 times per week, respectively, tocancer-bearing mice having a tumor grown 30 to 140 mm³ to which aBurkitt's lymphoma cell line Ramos had been transplanted.

FIG. 15 shows a content of aggregates measured by HPLC in amino-acidmodified antibodies of human anti-CD98 monoclonal antibodies C2IgG1NSafter the purification thereof.

FIG. 16A shows the binding of human anti-CD98 monoclonal antibodies K3and C2IgG1 as well as each amino-acid modified antibodies of C2IgG1 tohuman CD98/human LAT1-expressing L929 cell line.

FIG. 16B shows the binding of human anti-CD98 monoclonal antibodies K3and C2IgG1 as well as each amino-acid modified antibodies of C2IgG1 to ahuman colorectal cancer cell line (DLD-1), a Burkitt's lymphoma cellline (Ramos), a human colorectal cancer cell line (Colo205), and humanaortic endothelial cells (HAEC).

DETAILED DESCRIPTION OF THE INVENTION

Deposit of Microorganisms

Plasmid vectors C2IgG1/pCR4 and K3/pCR4 containing the nucleotidesequences coding for the variable region of the human antibody providedby the present invention were deposited on Mar. 14, 2006 to theInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology (Tsukuba Central 6, 1-1, Higashi1-chome, Tsukuba-shi, Ibaraki-ken, Japan) with the accession Nos. FERMBP-10551 (indication for identification: C2IgG1/pCR4) and FERM BP-10552(indication for identification: K3/pCR4), respectively.

Definitions

One-letter notations used for the description of amino acids in thepresent specification and Figures refer to the following respectiveamino acids: (G) glycine, (A) alanine, (V) valine, (L) leucine, (I)isoleucine, (S) serine, (T) threonine, (D) aspartic acid, (E) glutamicacid, (N) asparagine, (Q) glutamine, (K) lysine, (R) arginine, (C)cystein, (M) methionine, (F) phenylalanine, (Y) tyrosine, (W)tryptophan, (H) histidine, and (P) proline. One-letter alphabeticnotations for the designation of DNA are as follows: (A) adenine, (C)cytocine, (G) guanine, and (T) thymine.

CD98 and Monoclonal Antibody Thereagainst

CD98, to which the human monoclonal antibody and a functional fragmentthereof according to the present invention (hereinbelow abbreviated as“antibody according to the present invention,” unless otherwise noted)has a specific binding ability, is a type II transmembrane glycoproteinchain composed of 529 amino acid residues and is in the form of aheterodimer with a protein having an amino acid transporter activity onthe cell membrane, as described above. A preferred specific example ofthe protein having an amino acid transporter activity is LAT1. Further,in a preferred embodiment of the present invention, the CD98 is humanCD98. The primary structure of the human CD98 protein is known (SEQ IDNO: 66; GenBank/EMBL/DDBJ accession No. AB018010) and that of the humanLAT1 protein is also known (SEQ ID NO: 68; GenBank/EMBL/DDBJ accessionNo. AB018009).

The antibody according to the present invention has a specific bindingability to CD98 which is derived from a cell membrane of a cancer celland is in the form of a complex with a protein having an amino acidtransporter activity, while the antibody does not bind to normal humancells, for example, normal human vascular endothelial cells, normalhuman peripheral blood monocytes, or lymphocytes. Examples of the cancercells to which the antibody has a binding ability include cancer cellsconstituting colorectal cancer, lung cancer, breast cancer, prostaticcancer, melanoma, brain tumor, lymphoma, bladder cancer, pancreaticcancer, multiple myeloma, renal cell carcinoma, leukemia, T-celllymphoma, gastric cancer, pancreatic cancer, cervical cancer,endometrial cancer, ovarian cancer, esophageal cancer, liver cancer,head and neck squamous cell carcinoma, skin cancer, urinary tractcancer, prostatic cancer, chorionic carcinoma, pharyngeal cancer,laryngeal cancer, pleural tumor, arrhenoblastoma, endometrialhyperplasia, endometriosis, embryoma, fibrosarcoma, Kaposi's sarcoma,angioma, cavernous angioma, hemangioblastoma, retinoblastoma,astrocytoma, neurofibroma, oligodendroglial tumor, medulloblastoma,neuroblastoma, gliocystoma, rhabdomyoblastoma, glioblastoma, osteogenicsarcoma, leiomyosarcoma, thyroid sarcoma, Wilms tumor or the like, morespecifically, colorectal cancer cell lines (DLD-1, Colo205, SW480,SW620, LOVO, LS180, and HT29), a lung cancer cell line (H226), aprostate cancer cell line (DU145), a melanoma cell line (G361, SKMEL28,and CRL1579), a non-Hodgkin lymphoma cell line (Ramos), a bladder cancercell line (T24), a breast cancer cell line (MCF and MDA-MB-231), apancreatic cancer cell line (HS766T), a multiple myeloma cell line(IM9), an erythroblastic leukemia cell line (K562). The antibodyaccording to the present invention is advantageous in terms of thebinding ability to such a variety of cancer cells.

The specific binding ability of the antibody according to the presentinvention to cancer cells increases usefulness of the antibody accordingto the present invention. In other words, as described below, theantibody according to a preferred embodiment of the present inventionadvantageously binds only to cancer cells in order to significantlyinhibit the amino acid uptake into cells via LAT1, and the antibodyaccording to the present invention can be advantageously used, as atargeting agent, to bind to another drug and deliver the drug to cancercells.

Further, the antibody according to the present invention has ananti-tumor activity. The antibody according to a preferred embodiment ofthe present invention has a property of significantly inhibiting theamino acid uptake into cells via LAT1. Accordingly, the anti-tumoractivity of the antibody according to the present invention isconsidered to be attributable to giving a specific damage using animmune system by ADCC and CDC, as well as to inhibiting the amino aciduptake as in the above. In a more specific embodiment of the presentinvention, the antibody according to the present invention significantlyinhibits the amino acid uptake of bladder cancer cell line T24 cells.

In a preferred embodiment of the present invention, the antibodyaccording to the present invention has any pair of sequences of (a) SEQID NOs: 29 and 31, (b) SEQ ID NOs: 41 and 47, and (c) SEQ ID NOs: 43 and47 described below as a heavy chain variable region and a light chainvariable region thereof. Further, in another embodiment of the presentinvention, the antibody according to the present invention has, asvariable regions, sequences encoded by sequences contained in a plasmidvector K3/pCR4 (FERM BP-10552) or C2IgG1/pCR4 (FERM BP-10551) providedthat the sequence from a vector pCR4 is excluded. The amino acidsequences of the variable regions of the antibody according to thisembodiment are encoded by a BglII-BsiWI fragment (light chain variableregion) and a SalI-NheI fragment (heavy chain variable region), whichare obtained from any of the plasmid vectors described above and containno sequence from a vector pCR4.

The functional fragment of the antibody according to the presentinvention refers to a fragment of the antibody specifically binding tothe antigen to which the antibody according to the present inventionspecifically binds, and more specifically includes F(ab′)2, Fab′, Fab,Fv, disulphide-linked FV, Single-Chain FV (scFV) and polymers thereof,and the like (D. J. King., Applications and Engineering of MonoclonalAntibodies., 1998, T.J. International Ltd). These antibody fragments canbe obtained by a conventional method, for example, digestion of anantibody molecule by a protease such as papain, pepsin and the like, orby a known genetic engineering technique.

“Human antibody” used in the present invention refers to an antibodythat is an expression product of a human-derived antibody gene. Thehuman antibody can be obtained by administration of an antigen to atransgenic animal to which a human antibody locus has been introducedand which has an ability of producing human-derived antibody. An exampleof the transgenic animal includes a mouse, and a method of creating amouse capable of producing a human antibody is described in, forexample, WO 02/43478 pamphlet.

The antibody according to the present invention also includes amonoclonal antibody composed of a heavy chain and/or a light chain, eachhaving an amino acid sequence in which one or several amino acids aredeleted, substituted, or added in each amino acid sequence of a heavychain and/or a light chain constituting the antibody. Such a partialmodification (deletion, substitution, insertion, or addition) of anamino acid(s) can be introduced into the amino acid sequence of theantibody according to the present invention by partially modifying thenucleotide sequence encoding the amino acid sequence. The partialmodification of a nucleotide sequence can be introduced by an ordinarymethod using known site-specific mutagenesis (Proc Natl Acsd Sci USA.,1984, Vol 8, 15662; Sambrook et al., Molecular Cloning A LaboratoryManual (1989) Second edition, Cold Spring Harbor Laboratory Press).

In a preferred embodiment of the present invention, the antibodyaccording to the present invention is an antibody in which isoleucine atposition 117 of the light chain is substituted with another amino acidresidue, for example, methionine, asparagine, leucine or cystein.Preferred examples of such an antibody include those having, as a heavychain variable region and a light chain variable region, any pair ofsequences of (d) SEQ ID NOs: 43 and 77, (e) SEQ ID NOs: 43 and 79, (f)SEQ ID NOs: 43 and 81, and (g) SEQ ID NOs: 43 and 83.

The antibody according to the present invention includes an antibodyhaving any immunoglobulin class and subclass. In a preferred embodimentof the present invention, the antibody is an antibody of humanimmunoglobulin class and subclass, and preferred class and subclassesare immunoglobulin G (IgG), especially, IgG1, and a preferred lightchain is K.

Further, the antibody according to the present invention also includesan antibody converted into a different subclass by modification bygenetic engineering known by the person skilled in the art (for example,EP0314161). In other words, an antibody of a subclass different from theoriginal subclass can be obtained from a DNA encoding a variable regionof the antibody according to the present invention by geneticengineering technique.

ADCC refers to a cytotoxic activity that is induced by recognition of acell through binding to a constant region of an antibody via an Fcreceptor expressed on the surface of macrophages, NK cells, neutrophils,and the like and activation of the recognized cell. On the other hand,CDC refers to a cytotoxic activity caused by the complement systemactivated by binding of an antibody to an antigen. It has been revealedthat the strength of these activities differs depending on the subclassof antibody and the difference is due to a difference in the structureof a constant region of an antibody (Charles A. Janeway, et. al.Immunobiology, 1997, Current Biology Ltd/Garland Publishing Inc.).

Accordingly, for example, an antibody having a lower binding strength toan Fc receptor can be obtained by converting the subclass of theantibody according to the present invention to IgG2 or IgG4. On thecontrary, an antibody having a higher binding strength to an Fc receptorcan be obtained by converting the subclass of the antibody according tothe present invention into IgG1 or IgG3. When the above ADCC and CDCactivities are expected, the subclass of antibody is desirably IgG1.

When an antibody of a different subclass is converted into IgG1, IgG1can be prepared, for example, by isolating only a variable region froman antibody-producing hybridoma and introducing the variable region intoa vector containing the constant region of human IgG1, for example,N5KG1-Val Lark vector (IDEC Pharmaceuticals, N5KG1 (U.S. Pat. No.6,001,358)).

Further, it is possible to change a binding strength to an Fc receptorby modifying the amino acid sequence of the constant region of theantibody according to the present invention by genetic engineering, orby binding a constant region sequence having such a sequence (seeJaneway C A. Jr. and Travers P. (1997), Immunobiology, Third Edition,Current Biology Ltd./Garland Publishing Inc.), or to change a bindingstrength to a complement (see Mi-Hua Tao, et al., 1993, J. Exp. Med).For example, a binding strength to a complement can be changed bysubstituting proline with serine by mutating the sequence CCC encodingproline (P) at position 331 according to the EU Numbering System (seeSequences of proteins of immunological interest, NIH Publication No.91-3242) of the constant region of the heavy chain into the sequence TCCencoding serine (S).

For example, if the antibody according to the present invention byitself does not possess a cell death-inducing activity, an antibodyhaving antitumor activity due to antibody-dependent cell-mediatedcytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) via anFc receptor is desirable. However, when the antibody by itself has acell death-inducing activity, an antibody having a lower bindingstrength to an Fc receptor may be more desirable in some cases.

Considering immunosuppressants, an antibody having no ADCC activity orno CDC activity is desired, when sterically inhibiting only the bindingof T cells and antigen-presenting cells, and the like. In addition, whenan ADCC activity or a CDC activity may be a cause of toxicity, anantibody in which an activity causing toxicity is avoided by mutatingthe Fc region or changing the subclass may be desirable in some cases.

Considering above, the antibody according to the present invention maybe an antibody that can specifically damage cancer cells by ADCC or CDCby converting to another subclass through genetic engineering, ifnecessary.

In another preferred embodiment of the present invention, the antibodyaccording to the present invention preferably recognizes an epitopeconstituted by at least 8 consecutive or non-consecutive amino acidresidues in the amino acid sequence of human CD98 (SEQ ID NO: 66). In amore preferred embodiment of the present invention, the antibodyaccording to the present invention preferably has a binding ability topart of the region of amino acids 371 to 529 or part of the region ofamino acids 1 to 371 of the amino acid sequence of human CD98.

In another embodiment of the present invention, an antibody havingcross-reactivity to human CD98 and monkey CD98 is provided as theantibody according to the present invention. Such an antibody is assumedto recognize the same or a highly similar epitope structure in humanCD98 and monkey CD98, and has an advantage that various tests can beconducted in monkeys as experimental animals prior to clinical studiesin human.

Preparation of CD98 Antibody

The antibody according to the present invention can be produced by, forexample, the following method. Immunization is conducted by immunizingnon-human mammals such as mice, rabbits, goats, horses and the like withhuman CD98/human LAT1 or a part thereof, or a conjugate thereof with anappropriate substance (for example, bovine serum albumin) to enhanceantigenicity of an antigen, or cells on the surface of which humanCD98/human LAT1 is expressed in a large amount, in combination with animmunostimulant (Freund's Adjuvant, etc.), if required, or byadministering an expression vector incorporated with human CD98/humanLAT1 to the non-human mammals. The antibody according to the presentinvention can be obtained by preparing a hybridoma fromantibody-producing cells obtained from an immunized animal and myelomacells lacking autoantibody producing ability, cloning the hybridoma, andselecting a clone producing a monoclonal antibody exhibiting specificaffinity to the antigen used for immunization.

The method for preparing the antibody according to the present inventionwill be more specifically described in detail below, but the method forpreparing the antibody is not limited thereto, and, for example,antibody-producing cells other than spleen cells and myeloma can also beused.

As the antigen, a transformant that is obtained by incorporating a DNAencoding CD98 into an expression vector for animal cells and introducingthe expression vector to animal cells can be used.

Since CD98 forms a heterodimer with LAT1 on the cell surface of manycancer cells, an antibody that inhibits amino acid uptake by LAT1 isexpected to be obtained by incorporating a DNA encoding LAT1 into anexpression vector in a similar manner and using a transformant in whichCD98 and LAT1 are coexpressed as an antigen.

As the expression vector for animal cells, for example, vectors such aspEGF-N1 (manufactured by Becton Dickinson Bioscience Clontech) can beused. A vector for introducing an intended gene can be prepared bycleaving an insertion site by an appropriate restriction enzyme andlinking the human CD98 or human LAT1 cleaved by the same enzyme. Theprepared expression vector can be introduced into cells, for example,L929 cells (American Type Culture Collection No. CCL-1) as a host toprepare cells highly expressing human CD98 and human LAT1.

The methods for introducing a gene into a host are known and include,for example, any methods (for example, a method using a calcium ion, anelectroporation method, a spheroplast method, a lithium acetate method,a calcium phosphate method, a lipofection method and the like).

The transformed cells thus prepared can be used as an immunogen forpreparing a CD98 antibody. The expression vector itself can also be usedas an immunogen.

Human CD98 can be produced by appropriately using a method known in theart, such as a genetic recombination technique as well as a chemicalsynthesis method and a cell culture method based on the known nucleotidesequence or amino acid sequence of the CD98. The human CD98 protein thusobtained can also be used as an antigen to prepare a CD98 antibody. Apartial sequence of human CD98 can also be produced by a generecombination technique or a chemical synthesis in accordance with amethod known in the art described below, or produced by cleaving humanCD98 appropriately using a protein degradation enzyme, or the like.

The antigen obtained as described above can be used for immunization asdescribed below. Specifically, the prepared antigen is mixed with anappropriate substance for enhancing the antigenicity (for example,bovine serum albumin and the like) and an immunostimulant (Freundcomplete or incomplete adjuvant, and the like), as required, and usedfor immunization of a non-human mammal such as a mouse, rabbit, goat,and a horse. In addition, preferably, the antibody according to thepresent invention may be obtained as a human antibody using a non-humananimal that has an unrearranged human antibody gene and produces a humanantibody specific to the antigen by immunization. In this case, examplesof the animals producing human antibody include transgenic miceproducing human antibody described in the literature of Tomizuka et al.(Tomizuka et al., Proc. Natl. Acad. Sci. USA, 2000, Vol 97: 722).

A hybridoma secreting a monoclonal antibody can be prepared by a methodof Kohler and Milstein (Nature, 1975, Vol. 256: 495-497) or inaccordance with the method. In other words, a hybridoma is prepared bycell fusion between antibody producing cells contained in the spleen,lymph nodes, bone marrow, tonsil, or the like, preferably contained inthe lymph nodes or spleen, obtained from an animal immunized asdescribed above, and myeloma cells that are derived preferably from amammal such as a mouse, a rat, a guinea pig, hamster, a rabbit, a humanor the like and incapable of producing any autoantibody. Cell fusion canbe performed by mixing antibody-producing cells with myeloma cells in ahigh concentration solution of a polymer such as polyethylene glycol(for example, molecular weight of 1500 to 6000) usually at about 30 to40° C. for about 1 to 10 minutes. A hybridoma clone producing amonoclonal antibody can be screened by culturing a hybridoma on, forexample, a microtiter plate, and measuring reactivity of a culturesupernatant from wells in which the hybridoma is grown to animmunization antigen using an immunological method such as an enzymeimmunoassay (for example, ELISA), a radioimmunoassay, or a fluorescentantibody method.

A monoclonal antibody can be produced from a hybridoma by culturing thehybridoma in vitro and then isolating monoclonal antibodies from aculture supernatant. A monoclonal antibody can also be produced by ahybridoma by culturing the hybridoma in ascites or the like of a mouse,a rat, a guinea pig, a hamster, a rabbit, or the like in vivo andisolating the monoclonal antibody from the ascites. Further, arecombinant antibody can be prepared by a genetic recombinationtechnique by cloning a gene encoding a monoclonal antibody fromantibody-producing cells such as a hybridoma and the like, andincorporating the gene into an appropriate vector, introducing thevector to a host (for example, cells from a mammalian cell line, such asChinese hamster ovary (CHO) cells and the like, E. coli, yeast cells,insect cells, plant cells and others (P. J. Delves. ANTIBODY PRODUCTIONESSENTIAL TECHNIQUES., 1997, WILEY, R Shepherd and C. Dean., MonoclonalAntibodies., 2000, OXFORD UNIVERSITY PRESS, J. W. Goding, MonoclonalAntibodies: principles and practice., 1993, ACADEMIC PRESS). Further, bypreparing a transgenic bovine, goat, sheep or porcine in which a gene ofa target antibody is incorporated into an endogenous gene using atransgenic animal production technique, a large amount of a monoclonalantibody derived from the antibody gene may be obtained from the milk ofthe transgenic animal. When a hybridoma is cultured in vitro, thehybridoma is grown, maintained, and stored depending on the variousconditions such as properties of a cell species to be cultured,objectives of a study, and culture methods and the like, and the culturemay be conducted using a known nutritional medium or any nutritionalmediums induced and prepared from a known basic medium that are used toproduce a monoclonal antibody in a culture supernatant.

The produced monoclonal antibody can be purified by a method known inthe art, for example, by appropriate combination of chromatography usinga protein A column, ion-exchange chromatography, hydrophobicchromatography, ammonium sulfate salting-out, gel filtration, affinitychromatography, and the like.

Pharmaceutical Use of Antibody

The antibody according to the present invention can form a complex thatmay be used for the purpose of treatment such as drug delivery to cancercells, missile therapy, and the like, by conjugating the antibody with atherapeutic agent, because of the specific binding ability to CD98 whichis derived from the cell membrane of cancer cells and is in the form ofa complex with a protein having an amino acid transporter activity.

Examples of the therapeutic agent to be conjugated to the antibodyinclude, but not limited to, radionuclides such as iodine (¹³¹iodine:¹³¹I, ¹²⁵iodine: ¹²⁵I), yttrium (⁹⁰yttrium: ⁹⁰Y), indium (¹¹¹indium:¹¹¹In), technetium (^(99m)technetium: ^(99m)Tc) and the like (J. W.Goding. Monoclonal Antibodies: principles and practice., 1993, ACADEMICPRESS); bacterial toxins such as Pseudomonas exotoxin, diphtheria toxin,and ricin; and chemotherapeutic agents such as methotrexate, mitomycin,calicheamicin and the like (D. J. King., Applications and Engineering ofMonoclonal Antibodies., 1998, T.J. International Ltd, M. L. Grossbard.,Monoclonal Antibody-Based Therapy of Cancer., 1998, Marcel Dekker Inc),preferably a selenium compound inducing a radical production.

An antibody may be bound to a therapeutic agent via covalent bonding ornon-covalent bonding (for example, ion bonding). For example, thecomplex of the present invention can be obtained, using a reactive group(for example an amine group, a carboxyl group, or a hydroxy group) in amolecule or a coordination group, after binding to a more reactive groupor being converted into a reactive group, as required, by bringing anantibody into contact with a therapeutic agent having an functionalgroup capable of reacting with the reactive group to form bonding (inthe case of bacterial toxin or chemotherapeutic agent) or an ionic groupcapable of forming a complex with the coordination bond (in the case ofradionuclide). Alternatively, a biotin-avidin system can also beutilized for complex formation.

When the therapeutic agent is a protein or a peptide, a fusion proteinof the antibody and the protein or peptide can be produced by a geneticengineering technique.

Further, since the antibody according to the present invention has anantitumor activity, the antibody itself can be used as an anti-tumoragent. In addition, the antibody can be used as an active ingredient ofa pharmaceutical composition, especially a preventive or therapeuticagent for tumors.

Accordingly, the antibody or the pharmaceutical composition according tothe present invention can be applied to treatment or prevention ofvarious diseases or symptoms that may be attributable to the cellsexpressing human CD98/human LAT1. Examples of the disease or symptominclude various malignant tumors, and examples of the tumor includecolorectal cancer, lung cancer, breast cancer, prostatic cancer,melanoma, brain tumor, lymphoma, bladder cancer, pancreatic cancer,multiple myeloma, renal cell carcinoma, leukemia, T-cell lymphoma,gastric cancer, pancreatic cancer, cervical cancer, endometrial cancer,ovarian cancer, esophageal cancer, liver cancer, head and neck squamouscell carcinoma, skin cancer, urinary tract cancer, prostatic cancer,chorionic carcinoma, pharyngeal cancer, laryngeal cancer, pleural tumor,arrhenoblastoma, endometrial hyperplasia, endometriosis, embryoma,fibrosarcoma, Kaposi's sarcoma, angioma, cavernous angioma,hemangioblastoma, retinoblastoma, astrocytoma, neurofibroma,oligodendroglial tumor, medulloblastoma, neuroblastoma, gliocystoma,rhabdomyoblastoma, glioblastoma, osteogenic sarcoma, leiomyosarcoma,thyroid sarcoma, Wilms tumor and the like. The antibody according to thepresent invention may be applied not only to one tumor but multipletumors complicated together. The human monoclonal antibody according tothe present invention may be applied for prolongation of the life of apatient with primary local cancer. Further, the pharmaceuticalcomposition according to the present invention is allowed to actselectively on immunocompetent cells expressing CD98.

A medicament containing the antibody according to the present inventionor the antibody bound to a therapeutic agent is preferably provided as apharmaceutical composition.

Such a pharmaceutical composition contains a therapeutically effectiveamount of a therapeutic agent and is formulated into various forms fororal or parenteral administration. The therapeutically effective amountused herein refers to an amount that exhibits a therapeutic effect on agiven symptom in a given dosage regimen.

The composition according to the present invention may comprises, inaddition to the antibody, one or more physiologically acceptablepharmaceutical additives, for example, diluents, preservatives,solubilizers, emulsifiers, adjuvants, antioxidants, tonicity agents,excipients, and carriers. Further, the composition can be a mixture withother drugs such as other antibodies or antibiotics.

Examples of the appropriate carrier include, but not limited to,physiological saline, phosphate buffered physiological saline, phosphatebuffered physiological saline glucose solution and bufferedphysiological saline. Stabilizers such as amino acids, sugars,surfactants and the like and surface-adsorption inhibitors that areknown in the art may be contained.

As the form of the formulation, a formulation can be selected dependingon an object of the treatment and therapeutic regimen from formulationsincluding lyophilized formulations (that can be used afterreconstitution by addition of a buffered aqueous solution as describedabove), sustained-release formulations, enteric formulations,injections, drip infusions and the like.

A route of administration may be determined appropriately, and an oralroute as well as a parenteral route including intravenous,intramuscular, subcutaneous and intraperitoneal injections and drugdeliveries may be considered. Alternatively, a method in which thecomposition according to the present invention is brought into contactdirectly with an affected site of a patient may also be conducted.

The dose can be appropriately determined by animal studies and clinicalstudies, but in general, should be determined in consideration of acondition or severity, age, body weight, sex, and the like of a patient.In general, for oral administration, the dose is about 0.01 to 1000mg/day for adults, which may be administered once daily or divided intoseveral times a day. For parenteral administration, about 0.01 to 1000mg/dose can be administered by subcutaneous injection, intramuscularinjection or intravenous injection.

The present invention encompasses a preventive or therapeutic method ofthe diseases described above using the antibody or pharmaceuticalcomposition according to the present invention, and also encompasses useof the antibody according to the present invention for the manufactureof a preventive or therapeutic agent for the diseases described above.

In a preferred embodiment of the present invention, the antibodyaccording to the present invention is used as an ampoule containing asterile solution or suspension obtained by dissolving the antibody inwater or a pharmacologically acceptable solution. In addition, a sterilepowder formulation (preferably, a molecule of the present invention islyophilized) may be filled in an ampoule and reconstituted with apharmacologically acceptable solution at the time of use.

EXAMPLES

The present invention will be illustrated in more detail by thefollowing Examples, but the present invention is not limited to theembodiments described in these Examples.

Example 1 Preparation of Human CD98 or Human LAT1 Expression Vector

Polymerase chain reaction (PCR) was conducted using plasmid vectorspcDNA3.1-hCD98 and pcDNA3.1-hLAT1 containing DNA of human CD98 (hCD98,GenBank/EMBL/DDBJ accession no. AB018010; SEQ ID NO: 65) and human LAT1(hLAT1, GenBank/EMBL/DDBJ accession no. AB018009; SEQ ID NO: 67),respectively, as templates. In order to add the EcoRI sequence to the 5′end of the full length human CD98 cDNA and the NotI sequence and atermination codon to the 3′ end, primers 5′-CCG GAA TTC CCA CCA TGA GCCAGG ACA CCG AGG TGG ATA TGA-3′ (SEQ ID NO: 59) and 5′-AAG GAA AAA AGCGGC CGC TCA TCA GGC CGC GTA GGG GAA GCG GAG CAG CAG-3′ (SEQ ID NO: 60)were used and KOD-Plus DNA Polymerase (manufactured by Toyobo) andhCD98c DNA (about 20 ng) were used as templates to perform 30 cycles ofPCR at 94° C. for 15 seconds, at 55° C. for 30 seconds, and at 68° C.for 1 minute 30 seconds. The modified hCD98 sequence was isolated as anEcoRI-NotI fragment and ligated to a pTracer-EF/Bsd vector (manufacturedby Invitrogen) that had been cleaved by the same enzyme. The obtainedplasmid was used as a template and CD98 v2 U (5′-AGT CTC TTG CAA TCG GCTAAG AAG AAG AGC ATC CGT GTC ATT CTG-3′ (SEQ ID NO: 61)) primer and CD98v2 L (5′-CAG AAT GAC ACG GAT GCT CTT CTT CTT AGC CGA TTG CAA GAG ACT-3′(SEQ ID NO: 62)) primer were used to change A of positions of 591 and594 of hCD98 DNA (positions of 702 and 705 in SEQ ID NO: 65) to G. AnEcoRI-hCD98-NotI fragment was prepared from the obtained plasmid andligated to pEF6myc-His/Bsd (Invitrogen) vector that had been cleaved bythe same enzyme. The obtained plasmid was named as pEF6/hCD98.

In a similar manner, in order to add the EcoRI sequence to the 5′ end ofthe full length human LAT1 cDNA and the KpnI sequence to the 3′ end,primers 5′-CCG GAA TTC CCA CCA TGG CGG GTG CGG GCC CGA AGC GGC-3′ (SEQID NO: 63) and 5′-CGG GGT ACC GTC TCC TGG GGG ACC ACC TGC ATG AGC TTC-3′(SEQ ID NO: 64) were used and KOD-Plus DNA polymerase and hLAT1 cDNA(about 20 ng) were used as templates to perform 30 cycles of PCRreaction at 94° C. for 15 seconds; at 55° C. for 30 seconds; and at 68°C. for 1 minute 30 seconds. The modified hLAT1 sequence was isolated asan EcoRI-KpnI fragment and ligated to a pEGFP-N1 (manufactured byClontech) vector that had been cleaved by the same enzyme. Further, theobtained plasmid was isolated as an EcoRI-NotI fragment and ligated to apEF1V5His/Neo (manufactured by Invitrogen) vector that had been cleavedby the same enzyme. The obtained plasmid was named as pEF1/hLAT1-EGFP.

Example 2 Preparation of hCD98/hLAT1-Expressing Cells

hCD98/hLAT1-expressing cells were prepared by introducing the expressionvectors pEF6/hCD98 and pEF1/hLAT1-EGFP (hLAT1-E) prepared in Example 1to Colon 26 (CT26) cells and L929 cells (American Type CultureCollection No. CCL-1) using Lipofectamine and Plus reagent manufacturedby Invitrogen. The gene introduction was conducted in accordance withthe method described in the manual. The transgenic cells were culturedin a cell culture plate (6-well plate, manufactured by Becton Dickinson)at 37° C. in 5% carbonate gas for 24 hours and then cultured in aculture medium containing blasticidin (5 μg/mL) and G418 (500 μg/mL) inthe case of the CT26 cell line and a culture medium containingblasticidin (5 μg/mL) and G418 (1 mg/mL) in the case of the L929 cellline for further 3 days. hLAT1-E and CD98 positive cells were thenseparated by FACS Vantage using RPE fluorescently-labeled mouseanti-human CD98 antibody (Becton Dickinson, Ca. No. 556076).hCD98-expressing L929 cells or hLAT1-E-expressing L929 cells wereprepared in a similar manner.

Example 3 Preparation of Human Antibody-Producing Mice

Mice used for immunization have a genetic background of being homozygousfor disruption of both endogenous Ig heavy chain and K light chain andretaining a chromosome 14 fragment (SC20) containing a human Ig heavychain locus and a human IgK chain transgene (KCo5) simultaneously. Thesemice were prepared by crossing a mouse of line A having a human Ig heavychain locus and a mouse of line B having a human Ig K chain transgene.The mice of line A are homozygous for disruption of both endogenous Igheavy chain and K light chain and retain a chromosome 14 fragment (SC20)that is transmissible to progeny, and are described, for example, in thereport of Tomizuka, et al. (Tomizuka. et al., Proc Natl Acad Sci USA,2000, Vol 97: 722). The mice of line B are transgenic mice, which arehomozygous for disruption of both endogenous Ig heavy chain and K lightchain and retain a human Ig K chain transgene (KCo5), and are described,for example, in the report of Fishwild, et al. (Nat. Biotechnol., 1996,Vol 14: 845).

Progeny mice obtained by crossing a male mouse of line

A and a female mouse of line B or a female mouse of line A and a malemouse of line B were analyzed by the method described in the report ofTomizuka (Tomizuka et al., Proc Natl Acad Sci USA, 2000, Vol 97: 722),and individuals (human antibody-producing mice) for which human Ig heavychain and K light chain were detected simultaneously in serum werescreened (Ishida & Lonberg, IBC&apos;s 11th Antibody Engineering,Abstract, 2000; Ishida, I. et al., Cloning & Stem Cells 4, 85-96 (2002))and used in the following immunization experiments. In the immunizationexperiments, mice and the like having altered genetic backgrounds of theabove mice were also used (Ishida Isao (2002), Jikken Igaku, 20,6846851).

Example 4 Preparation of Human Monoclonal Antibodies

Monoclonal antibodies were prepared in accordance with a general methodas described in, for example, “Introduction of Experimental Protocolsfor Monoclonal Antibody” (Monoclonal Antibody Jikken Sosa Nyumon,written by Tamie ANDO et al., KODANSHA, 1991).

As an immunogen hCD98/hLAT1, the hCD98/hLAT1-E-expressing CT26 cellsprepared in Example 2 and human colorectal cancer cell line Colo205cells for which expression of hCD98 was confirmed were used.

As animals for immunization, the human antibody-producing mice producinghuman immunoglobulin that had been prepared in Example 3 were used.

When the hCD98/hLAT1-E-expressing CT26 cells were used, 5×10⁶ cells weremixed with RIBI adjuvant (manufactured by Corixa) and givenintraperitoneally for primary immunization. On days 7 and 24 after theprimary immunization, 5×10⁶ cells/mouse were given intraperitoneally forbooster immunization. The cells were further immunized in the samemanner 3 days prior to acquisition of spleen cells described below.

When Colo205 cells were used, 5×10⁶ cells were given intraperitoneallyfor primary immunization. On day 14 after the primary immunization,5×10⁶ cells/mouse were given intraperitoneally for booster immunization,and spleen cells described below were obtained 3 days later.

The spleen was obtained surgically from the immunized mice, and thespleen cells recovered were mixed with mouse myeloma SP2/0 (ATCC No.CRL1581) cells at a ratio of 5:1 and the cells were fused usingpolyethylene glycol 1500 (manufactured by Roche) as a fusing agent toprepare a large number of hybridomas. The hybridomas were cultured in aHAT-containing DMEM medium (manufactured by Gibco) containing 10% fetalcalf serum (FCS) and hypoxanthine (H), aminopterin (A), and thymidine(T) for screening. Single clones were obtained using a HT-containingDMEM medium by limiting dilution. A 96-well microtiter plate(manufactured by Becton Dickinson) was used for culturing. Selection(screening) for hybridoma clones producing an intended human monoclonalantibody and characterization of the human monoclonal antibodiesproduced by the respective hybridomas were conducted by measurement witha fluorescence-activated cell sorter (FACS) described below.

Screening for human monoclonal antibody-producing hybridomas wasconducted as described below. In other words, 200 or more hybridomasproducing human monoclonal antibodies, which contained a humanimmunoglobulin μ chain (hIgμ), a γ chain (hIgγ), and a humanimmunoglobulin light chain κ (hIgκ) and had specific reactivity tohCD98/hLAT1-E-expressing CT26 cells, were obtained by the FACS analysisdescribed below.

In Examples in the present specification, the hybridoma clones thatproduced each of the human monoclonal antibodies are named using thesymbols in Tables and Figures showing the results. The followinghybridoma clones represent single clones: 4-35-14 (C2), 4-32-9 (K3),7-95-8, 10-60-7, 3-69-6, 5-80-1 (for the above clones, the immunogen ishCD98/hLAT1-E-expressing CT26 cells); and 1-40-1 (for the clone, theimmunogen is Colo205 cells).

Example 5 Identification of Subclasses of Each of the MonoclonalAntibodies

The subclass of each of the monoclonal antibodies obtained in Example 4was identified by FACS analysis. 2×10⁶/mL of Colo205 cells weresuspended in a Staining Buffer (SB) of PBS containing 1 mM EDTA, 0.1%NaN₃, and 5% FCS. The cell suspension was dispensed in a 96-wellround-bottomed plate (manufactured by Becton Dickinson) at 50 μL/well.Further, the culture supernatant (50 μL) of the hybridoma cultured inExample 4 was added thereto, and the mixture was stirred, allowed toreact at ice temperature for 30 minutes, and then centrifuged (2000 rpm,4° C., 2 minutes) to remove the supernatant. After the pellets werewashed once with 100 μL/well of SB, an FITC fluorescently-labeled rabbitanti-human Igμ F(ab′)₂ antibody (manufactured by Dako Cytomation)diluted 50 time with SB, or an RPE fluorescently-labeled goat anti-humanIgγ F(ab′)₂ antibody (manufactured by SuthernBiotech) diluted 200 timeswith SB, or an RPE fluorescently-labeled rabbit anti-human Igκ F(ab′)₂antibody (manufactured by Dako Cytomation) diluted 200 times with SB wasadded thereto, and the mixture was allowed to react at ice temperaturefor 30 minutes. After washing once with SB, the cells were suspended in300 μL of SB and a fluorescence intensity indicating antibody bindingwas measured with an FACS (FACSCan, manufactured by Becton Dickinson).The results for parts of the obtained antibodies are shown in Table 1.For the C2, the heavy chain was μ chain and the light chain was κ chain,and for all of the K3, 3-69-6, 7-95-8, 10-60-7, 1-40-1, and 5-80-1, theheavy chain was γ chain and the light chain was κ chain.

TABLE 1 Subclass of antibodies Clone Light chain Heavy chain K3 Human κHuman γ C2 Human κ Human μ 1-40-1 Human κ Human γ 3-69-6 Human κ Human γ7-95-8 Human κ Human γ 10-60-7 Human κ Human γ 5-80-1 Human κ Human γ

Example 6 Preparation of Genes Encoding Monoclonal Antibodies andConstruction of Recombinant Antibody-Expression Vectors

Cloning of the genes of the respective antibodies C2, K3, 7-95-8,10-60-7, 3-69-6 and 1-40-1 and construction of expression vectors wereconducted in accordance with the methods described below.

(1) cDNA Cloning of Antibody Genes and Preparation of Expression Vectors

The hybridoma was cultured in a DMEM medium (manufactured by Gibco)containing 10% FCS, the cells were collected by centrifugation, and thenISOGEN (manufactured by Nippon Gene) was added to extract total RNA inaccordance the instruction manual. Cloning of the variable region of theantibody cDNAs was conducted using a SMART RACE cDNA amplification Kit(manufactured by Clontech) in accordance with the attached instructionmanual.

The 1st strand cDNA was prepared using 5 μg of the total RNA as atemplate.

(a) Synthesis of 1st Strand cDNA

A reaction solution having a composition of 5 μg/3 μL of the total RNA,1 μL of 5′CDS, and 1 μL of SMART oligo was incubated at 70° C. for 2minutes, then 2 μL of 5× buffer, 1 μL of DTT, 1 μL of DNTP mix, and 1 μLof Superscript II were added, and then the resultant mixture wasincubated at 42° C. for 15 hours. After 100 μL of Tricine Buffer wasadded, the resultant mixture was incubated at 72° C. for 7 minutes toobtain 1st strand cDNA.

(b) Amplification by PCR of Heavy Chain Genes and Light Chain Genes andConstruction of Recombinant Antibody Expression Vectors

KOD-Plus cDNA of Toyobo was used for amplification of cDNA.

A reaction solution having a composition of 15 μL of cDNA, 5 μL of10×KOD-Plus Buffer, 5 μL of dNTP mix, 1 μL of KOD-Plus, 3 μL of 25 mMMgSO₄, primer 1, and primer 2 was prepared in a final volume of 50 μLwith double distilled water and subjected to PCR.

Regarding K3, 1-40-1, 3-69-6, and C2, experimental examples arespecifically shown below.

K3

For amplification of the light chain, UMP and an hk-2 (5′-GTT GAA GCTCTT TGT GAC GGG CGA GC-3′ (SEQ ID NO: 1)) primer were used and a cycleof 94° C. for 5 seconds and 72° C. for 3 minutes was repeated 5 times,then a cycle of 94° C. for 5 seconds, 70° C. for 10 seconds, and 72° C.for 3 minutes was repeated 5 times, and further a cycle of 94° C. for 5seconds, 68° C. for 10 seconds, and 72° C. for 3 minutes was repeated 25times. Further, 1 μL of a 5-time diluted solution of this reactionsolution was used as a template, NUMP and a hk5 (5′-AGG CAC ACA ACA GAGGCA GTT CCA GAT TTC-3′ (SEQ ID NO: 2)) primer were used, and a cycle of94° C. for 15 seconds, 60° C. for 30 seconds, and 68° C. for 1 minutewas repeated 30 times. This reaction solution was subjected to 2%agarose gel electrophoresis, and the amplified PCR product was purifiedwith a QIA quick gel extraction kit (manufactured by Quiagen). Thepurified PCR product was ligated to a pCR4Blunt-TOPO vector(manufactured by Invitrogen) for subcloning in accordance with theattached instruction manual. T3 (5′-ATT AAC CCT CAC TAA AGG GA-3′ (SEQID NO: 3)) and the hk5 were then used as primers to determine thenucleotide sequence. Based on the sequence information, DNPL15Bglp(5′-AGA GAG AGA GAT CTC TCA CCA TGG AAG CCC CAG CTC AGC TTC TCT-3′ (SEQID NO: 4)) was synthesized. The light chain gene subcloned using thepCR4Blunt-TOPO vector was used as a template, the DNPL15Bglp and a 202LR(5′-AGA GAG AGA GCG TAC GTT TAA TCT CCA GTC GTG TCC CTT GGC-3′ (SEQ IDNO: 5)) primer were used, and a cycle of 94° C. for 15 seconds, 55° C.for 30 seconds, and 68° C. for 1 minute was repeated 30 times. Thisreaction solution was subjected to 2% agarose gel electrophoresis, and afragment of about 400 bp was purified by a QIAquick gel extraction kit(manufactured by Quiagen). The amplified light chain cDNA fragment wasdigested with BglII and BsiWI and the digested product was introducedinto an N5KG1-Val Lark vector (IDEC Pharmaceuticals, a modified vectorof N5KG1 (U.S. Pat. No. 6,001,358)) that had been cleaved by the sameenzymes. The vector thus obtained was named N5KG1-Val K3L.

For amplification of the heavy chain, UMP and an IgG1p (5′-TCT TGT CCACCT TGG TGT TGC TGG GCT TGT G-3′ (SEQ ID NO: 6)) primer were used, and acycle of 94° C. for 5 seconds and 72° C. for 3 minutes was repeated 5times, then a cycle of 94° C. for 5 seconds, 70° C. for 10 seconds, and72° C. for 3 minutes was repeated 5 times, further a cycle of 94° C. for5 seconds, 68° C. for 10 seconds, and 72° C. for 3 minutes was repeated25 times. Further, 1 μL of a 5-time diluted solution of this reactionsolution was used as a template, NUMP and IgG2p (5′-TGC ACG CCG CTG GTCAGG GCG CCT GAG TTC C-3′ (SEQ ID NO: 7)) were used, and a cycle of 94°C. for 15 seconds, 60° C. for 30 seconds, and 68° C. for 1 minute wasrepeated 30 times. This reaction solution was subjected to 2% agarosegel electrophoresis, and the amplified PCR product was purified by theQIAquick gel extraction kit. The purified PCR product was ligated to thepCR4Blunt-TOPO vector for subcloning. T3 and hh2 (5′-GCT GGA GGG CAC GGTCAC CAC GCT G-3′ (SEQ ID NO: 8)) were then used as primers to determinethe nucleotide sequence. Based on the sequence information, K3HcSalI(5′-AGA GAG AGA GGT CGA CCA CCA TGG GGT CAA CCG CCA TCC TCG CCC TCCTC-3′ (SEQ ID NO: 9)) was synthesized. The heavy chain gene subclonedusing the pCR4Blunt-TOPO vector was used as a template, K3HcSalI andF24HNhe (5′-AGA GAG AGA GGC TAG CTG AGG AGA CGG TGA CCA GGG TTC-3′ (SEQID NO: 10)) were used, and a cycle of 94° C. for 15 seconds, 55° C. for30 seconds, and 68° C. for 1 minute was repeated 25 times. This reactionsolution was subjected to 2% agarose gel electrophoresis, and a fragmentof about 450 bp was purified by the QIAquick gel extraction kit. Theamplified heavy chain cDNA fragment was digested with SalI and NheI, andthe digested product was introduced into the N5KG1-Val K3L that had beencleaved by the same enzymes. The DNA nucleotide sequence of the insertedportion was determined and it was confirmed that the sequence that hadbeen amplified by PCR and inserted was identical to the gene sequenceused as a template. The obtained vector was named N5KG1-Val K3IgG1.Whether or not a recombinant K3 antibody obtained by introducing theN5KG1-Val K3IgG1 into FreeStyle293 cells described below was identicalto the antibody derived from a K3 hybridoma was confirmed by determiningthe binding activity to the hCD98/hLAT1-expressing cell line.

1-40-1

For amplification of the heavy chain, UMP and an IgG1p primer were used,and a cycle of 94° C. for 5 seconds and 72° C. for 3 minutes wasrepeated 5 times, then a cycle of 94° C. for 5 seconds, 70° C. for 10seconds, and 72° C. for 3 minutes was repeated 5 times, further a cycleof 94° C. for 5 seconds, 68° C. for 10 seconds, and 72° C. for 3 minuteswas repeated 25 times. Further, 1 μL of a 5-time diluted solution ofthis reaction solution was used as a template, NUMP and an IgG2p primerwere used, and a cycle of 94° C. for 15 seconds, 60° C. for 30 seconds,and 68° C. for 1 minute was repeated 30 times. This reaction solutionwas subjected to 0.8% agarose gel electrophoresis, and the amplified PCRproduct was purified by the QIAquick gel extraction kit. The purifiedPCR product was ligated to the pCR4Blunt-TOPO vector for subcloning. T3and hh2 were then used as primers to determine the nucleotide sequence.Based on the sequence information, 205HP5SalI (5′-AGA GAG AGA GGT CGACCA CCA TGG AGT TTG GGC TGA GCT GGG TTT-3′ (SEQ ID NO: 11)) wassynthesized, and the heavy chain gene subcloned using the pCR4Blunt-TOPOvector was used as a template, 205HP5SalI and the F24Hnhe primer wereused, and a cycle of 94° C. for 15 seconds, 55° C. for 30 seconds, and68° C. for 1 minute was repeated 25 times. This reaction solution wassubjected to 2% agarose gel electrophoresis, and a fragment of about 450bp was purified by the QIAquick gel extraction kit. The amplified heavychain cDNA fragment was digested with SalI and NheI and the digestedproduct was introduced into the N5KG1-Val Lark vector that had beencleaved by the same enzymes. The obtained vector was named N5KG1-Val1-40-1H.

For amplification of the light chain, UMP and the hk-2 primer were used,and a cycle of 94° C. for 5 seconds and 72° C. for 3 minutes wasrepeated 5 times, then a cycle of 94° C. for 5 seconds, 70° C. for 10seconds, and 72° C. for 3 minutes was repeated 5 times, further a cycleof 94° C. for 5 seconds, 68° C. for 10 seconds, and 72° C. for 3 minuteswas repeated 25 times. Further, 1 μL of a 5-time diluted solution ofthis reaction solution was used as a template, NUMP and the hk5 wereused, and a cycle of 94° C. for 15 seconds, 60° C. for 30 seconds, and68° C. for 1 minute was repeated 30 times. This reaction solution wassubjected to 2% agarose gel electrophoresis, and the amplified PCRproduct was purified by the QIAquick gel extraction kit. The purifiedPCR product was ligated to the pCR4Blunt-TOPO vector for subcloning. T3and hk5 were then used as primers to determine the nucleotide sequence.Based on the sequence information, A27RN202 (5′-AGA GAG AGA GCG TAC GTTTGA TTT CCA CCT TGG TCC CTT GGC-3′ (SEQ ID NO: 12)) was synthesized, andthe light chain gene subcloned using the pCR4Blunt-TOPO vector was usedas a template, the DNPL15Bglp and the A27RN205 were used, and a cycle of94° C. for 15 seconds, 55° C. for 30 seconds, and 68° C. for 1 minutewas repeated 25 times. This reaction solution was subjected to 2%agarose gel electrophoresis, and a fragment of about 400 bp was purifiedby the QIAquick gel extraction kit. The amplified light chain cDNAfragment was digested with BglII and BsiWI and the digested product wasintroduced into the N5KG1-Val 1-40-1H vector that had been cleaved bythe same enzymes. The DNA nucleotide sequence of the inserted portionwas determined and it was confirmed that the sequence that had beenamplified by PCR and inserted was identical to the gene sequence used asa template. The obtained vector was named N5KG1-Val 1-40-1IgG1. Whetheror not a recombinant 1-40-1 antibody obtained by introducing theN5KG1-Val 1-40-1IgG1 into the FreeStyle293 cells described below wasidentical to the antibody derived from a 1-40-1 hybridoma was confirmedby determining the binding activity to the hCD98/hLAT1-expressing cellline.

3-69-6

For amplification of the light chain, UMP and the hk-2 primer were used,a cycle of 94° C. for 15 seconds and 72° C. for 3 minutes was repeated 5times, then a cycle of 94° C. for 15 seconds, 70° C. for 10 seconds, and72° C. for 3 minutes was repeated 5 times, further a cycle of 94° C. for15 seconds, 68° C. for 15 seconds, and 72° C. for 3 minutes was repeated25 times. Further, 2 μL of a 5-time diluted solution of this reactionsolution was used as a template, NUMP and the hk5 were used, and a cycleof 94° C. for 15 seconds, 60° C. for 30 seconds, and 68° C. for 1 minutewas repeated 30 times. This reaction solution was subjected to 0.8%agarose gel electrophoresis, and the amplified PCR product was purifiedby the QIAquick gel extraction kit. The purified PCR product was ligatedto the pCR4Blunt-TOPO vector for subcloning. A M13Foward(−20) primer(5′-GTA AAA CGA CGG CCA G-3′ (SEQ ID NO: 13)), a M13 Reverse primer(5′-CAG GAA ACA GCT ATG AC-3′ (SEQ ID NO: 14)), and the hk5 (5′-AGGCACACA ACA GAG GCAG TTCCAGA TTT C-3′ (SEQ ID NO: 2)) were then used asprimers to determine the nucleotide sequence. Based on the sequenceinformation, A27_F (5′-AGA GAG AGA GAT CTC TCA CCA TGG AAA CCC CAGCGCAGC TTC TCT TC-3′ (SEQ ID NO: 15)) and 39_(—)20_L3Bsi (5′-AGA GAG AGAGCG TAC GTT TGA TCT CCA GCT TGG TCC CCT G-3′ (SEQ ID NO: 16)) weresynthesized. The light chain gene subcloned using the pCR4Blunt-TOPOvector was used as a template, the A27_F and the 39_(—)20_L3Bsi wereused, a cycle of 94° C. for 30 seconds, 55° C. for 30 seconds, and 68°C. for 1 minute was repeated 25 times. This reaction solution wassubjected to 0.8% agarose gel electrophoresis, and a fragment of about400 bp was purified by the QIAquick gel extraction kit. The amplifiedlight chain cDNA fragment was digested by BglII and BsiWI and thedigested product was introduced into the N5KG1-Val Lark vector that hadbeen cleaved by the same enzymes. The obtained vector was namedN5KG1-Val 3-69-6L.

For amplification of the heavy chain, UMP and the IgG1p (5′-TCT TGT CCACCT TGG TGT TGC TGG GCT TGT G-3′ (SEQ ID NO: 6)) primer were used, acycle of 94° C. for 15 seconds, and 72° C. for 3 minutes was repeated 5times, then a cycle of 94° C. for 15 seconds, 70° C. for 10 seconds, and72° C. for 3 minutes was repeated 5 times, further a cycle of 94° C. for15 seconds, 68° C. for 15 seconds, and 72° C. for 3 minutes was repeated30 times. Further, 2 μL of a 5-time diluted solution of this reactionsolution was used as a template, NUMP and IgG2p (IgG1.3.4)(5′-TGC ACGCCG CTG GTCAGG GCG CCT GAG TTC C-3′ (SEQ ID NO: 7)) were used, a cycleof 94° C. for 30 seconds, 55° C. for 30 seconds, and 68° C. for 1 minutewas repeated 25 times. This reaction solution was subjected to 0.8%agarose gel electrophoresis, and the amplified PCR product was purifiedby the QIAquick gel extraction kit. The purified PCR product was ligatedto the pCR4Blunt-TOPO vector for subcloning. The M13F, M13R, and IgG2pwere then used as primers to determine the nucleotide sequence. Based onthe sequence information, Z3HP5Sal (5′-AGA GAG AGA GGT CGA CCCACCATG GACTGG AGCATC CTT TT-3′ (SEQ ID NO: 17)) and F24HNhe (5′-AGA GAG AGA GGCTAG CTG AGG AGA CGG TGA CCA GGG TTC-3′ (SEQ ID NO: 10)) weresynthesized. The heavy chain gene subcloned using the pCR4Blunt-TOPOvector was used as a template, the Z3HP5SalF and the F24HNhe were used,a cycle of 94° C. for 30 seconds, 55° C. for 30 seconds, and 68° C. for1 minute second was repeated 25 times. This reaction solution wassubjected to 0.8% agarose gel electrophoresis, and a fragment of about450 bp was purified by the QIAquick gel extraction kit. The amplifiedheavy chain cDNA fragment was digested with SalI and NheI and thedigested product was introduced into the N5KG1-Val 3-69-6L vector thathad been cleaved by the same enzymes. The DNA nucleotide sequence of theinserted portion was determined and it was confirmed that the sequencethat had been amplified by PCR and inserted was identical to the genesequence used as a template. The obtained vector was named N5KG1-Val3-69-6IgG1. Whether or not a recombinant 3-69-6 antibody obtained bytransfecting the N5KG1-Val 3-69-6IgG1 into the FreeStyle293 cellsdescribed below was identical to the antibody derived from a 3-69-6hybridoma was confirmed by determining the binding activity to thehCD98/hLAT1-expressing cell line.

C2IgG1

Since the subclass of the hybridoma-producing C2 is IgM, a C2 antibodyvariable region (C2 IgG1) was isolated by PCR using a primer that wasdesigned to contain a variable region assumed from IgG derived from thesame germ line.

For amplification of the light chain, UMP and the hk-2 primer were used,a cycle of 94° C. for 5 seconds, and 72° C. for 3 minutes was repeated 5times, then a cycle of 94° C. for 5 seconds, 70° C. for 10 seconds, and72° C. for 3 minutes was repeated 5 times, further a cycle of 94° C. for5 seconds, 68° C. for 10 seconds, and 72° C. for 3 minutes was repeated25 times. Further, 1 μL of a 5-time diluted solution of this reactionsolution was used as a template, NUMP and the hk5 were used, and a cycleof 94° C. for 15 seconds, 60° C. for 30 seconds, and 68° C. for 1 minutewas repeated 30 times. This reaction solution was subjected to 0.8%agarose gel electrophoresis, and the amplified PCR product was purifiedby the QIAquick gel extraction kit. The purified PCR product was ligatedto the pCR4Blunt-TOPO vector for subcloning. The M13F, M13R, and hk5were then used as primers to determine the nucleotide sequence. Based onthe sequence information, C2-1 Lc Bgl II F (5′-AGA GAG AGA GAT CTC TCACCA TGG AAA CCC CAG CGCAGC TTC TCT TC 3′ (SEQ ID NO: 18)) and C2-1 LcBsiWI R (5′-AGA GAG AGA GCG TAC GTT TGA TAT CCA CTT TGG TCC CAG GG-3′(SEQ ID NO: 19)) were synthesized. The light chain gene subcloned usingthe pCR4Blunt-TOPO vector was used as a template, the C2-1 Lc Bgl II Fand the C2-1 Lc BsiWI R were used, and a cycle of 94° C. for 15 seconds,60° C. for 30 seconds, and 68° C. for 1 minute was repeated 25 times.This reaction solution was subjected to 0.8% agarose gelelectrophoresis, and a fragment of about 400 bp was purified by theQIAquick gel extraction kit. The amplified light chain cDNA fragment wasdigested with BglII and BsiWI and the digested product was introducedinto the N5KG1-Val Lark vector that had been cleaved by the sameenzymes. The obtained vector was named N5KG1-Val C2L.

For amplification of the heavy chain, UMP and the M655R (5′-GGC GAA GACCCG GAT GGC TAT GTC-3′ (SEQ ID NO: 20)) primer were used, and a cycle of94° C. for 15 seconds, 60° C. for 30 seconds, and 68° C. for 1 minutewas repeated 30 times. Further, 1 μL of a 5-time diluted solution ofthis reaction solution was used as a template, NUMP and the M393R(5′-AAA CCC GTG GCC TGG CAG ATG AGC-3′ (SEQ ID NO: 21)) were used, and acycle of 94° C. for 15 seconds, 60° C. for 30 seconds, and 68° C. for 1minute was repeated 30 times. This reaction solution was subjected to0.8% agarose gel electrophoresis, and the amplified PCR product waspurified by the QIAquick gel extraction kit. The purified PCR productwas ligated to the pCR4Blunt-TOPO vector for subcloning. The M13Foward(−20) primer (5′-GTA AAA CGA CGG CCA G-3′ (SEQ ID NO: 13)), the M13Reverse primer (5′-CAG GAA ACA GCT ATG AC-3′ (SEQ ID NO: 14)), and theM393R were then used as primers to determine the nucleotide sequence.Based on the sequence information, C2hcSalIF (5′-AGA GAG AGA GGT CGA CCACCA TGA AGCACC TGT GGT TCT TCC TCC TGC T-3′ (SEQ ID NO: 22)) andC2hcNheI (5′-AGA GAG AGA GGC TAG CTG AGG AGA CGG TGA CCA GGG TTC CCTGG-3′ (SEQ ID NO: 58)) were synthesized. The heavy chain gene subclonedusing the pCR4Blunt-TOPO vector was used as a template, C2hcSalIF andC2hcNheI were used, a cycle of 94° C. for 15 seconds, 60° C. for 30seconds, and 68° C. for 30 seconds was repeated 25 times. This reactionsolution was subjected to 0.8% agarose gel electrophoresis, and afragment of about 450 bp was purified by the QIAquick gel extractionkit. The amplified heavy chain cDNA fragment was digested with SalI andNheI and the digested product was introduced into the N5KG1-Val C2Lvector that had been cleaved by the same enzymes. The DNA nucleotidesequence of the inserted portion was determined and it was confirmedthat the sequence that had been amplified by PCR and inserted wasidentical to the gene sequence used as a template. The obtained vectorwas named N5KG1-Val C2IgG1.

C2IgG1NS

The frame region of the heavy chain in the C2IgG1 gene cloned asdescribed above contained a mutation that is not observed in theoriginal germ line. A C2 variable region sequence having a sequence ofthe original germ line was thus isolated by the method described below.

The vector N5KG1-Val C2IgG1 obtained above was used as a template andthe C2hc NS F (5′-CGT CCA AGA ACC AGT TCT CCC TGA AGC TGA-3′ (SEQ ID NO:23)) primer and the C2hc NS R (5′-TCA GCT TCA GGG AGA ACT GGT TCT TGGACG-3′ (SEQ ID NO: 24)) primer were used to replace G and T at positions290 and 299 of the C2 antibody heavy chain with A and C, respectively,to prepare N5KG1-Val C2IgG1NS. Whether or not the recombinant C2IgG1 andC2IgG1NS antibodies that were obtained by introducing the N5KG1-ValC2IgG1 and the N5KG1-Val C2IgG1NS into the FreeStyle293 cells describedbelow, respectively, had the same specificity as that of the IgMantibody derived from the C2 hybridoma was confirmed by determination ofthe binding activity to the hCD98/hLAT1-expressing cell line. Thebinding activities of the C2IgG1 and the C2IgG1NS were almost the same.

C2IgμG1

Since the forms of binding sites of the heavy chain and the light chainmight differ from those of the original IgM when the method used in theabove C2IgG1 was used, the sequence conversion described below wasconducted. In other words, 26 amino acids contiguous in the variableregion side to a common sequence (GCL sequence) in the CH1 constantregion of the γ chain of IgG and the μ chain of IgM were used as a μchain sequence and all amino acids in the constant region side of theGCL sequence was converted into γ chain (C2 IgμG1). The above sequenceconversion was conducted by the method described below.

For amplification of the heavy chain of C2 cDNA, UMP and the M655Rprimer were used, and a cycle of 94° C. for 15 seconds, 60° C. for 30seconds, and 68° C. for 1 minute was repeated 30 times. Further, 1 μL ofa 5-time diluted solution of this reaction solution was used as atemplate, NUMP and the M393R were used, and a cycle of 94° C. for 15seconds, 60° C. for 30 seconds, and 68° C. for 1 minute was repeated 30times. This reaction solution was subjected to 0.8% agarose gelelectrophoresis, and the amplified PCR product was purified by theQIAquick gel extraction kit. The purified PCR product was ligated to thepCR4Blunt-TOPO vector for subcloning. The M13Foward(−20) primer (5′-GTAAAA CGA CGG CCA G-3′ (SEQ ID NO: 13)), the M13 Reverse primer (5′-CAGGAA ACA GCT ATG AC-3′ (SEQ ID NO: 14)), and the M393R were then used asprimers to determine the nucleotide sequence. Based on the sequenceinformation, C2hcSalIF (5′-AGA GAG AGA GGT CGA CCA CCA TGA AGCACC TGTGGT TCT TCC TCC TGC T-3′ (SEQ ID NO: 22)) and Mu-GCL-Gamma L(5′-CAC CGGTTC GGG GAA GTA GTC CTT GAC GAG GCAGCA AAC GGC CAC GCT GCT CGT-3′ (SEQID NO: 25)) were synthesized. The heavy chain gene subcloned using thepCR4Blunt-TOPO vector was used as a template, the C2hcSalIF and theMu-GCL-Gamma L were used, and a cycle of 94° C. for 15 seconds, 60° C.for 30 seconds, and 68° C. for 1 minute was repeated 25 times. Thisreaction solution was subjected to 0.8% agarose gel electrophoresis, andthe PCR amplification product was purified by the QIAquick gelextraction kit. This PCR amplification product was named C2Vμ. TheN5KG1-Val Lark vector was then used as a template, Mu-GCL-Gamma U(5′-ACG AGCAGC GTG GCC GTT GGC TGC CTC GTCAAG GAC TAC TTC CCC GAA CCGGTG-3′ (SEQ ID NO: 26)) and hIgG1 BamHI L (5′-CGC GGA TCC TCA TCA TTTACC CGG AGA CAG GGA GAG GCT-3′ (SEQ ID NO: 27)) were used, a cycle of94° C. for 15 seconds, 60° C. for 30 seconds, and 68° C. for 90 secondswas repeated 25 times. This reaction solution was subjected to 0.8%agarose gel electrophoresis, and the PCR amplification product waspurified by the QIAquick gel extraction kit. This PCR amplificationproduct was named Cγ1. Each 5 μL of 3-time diluted solutions of the C2Vμand the Cγ1 was placed, and a cycle of 94° C. for 15 seconds, 55° C. for30 seconds, and 68° C. for 2 minutes was repeated 3 times in the absenceof a primer. This reaction solution was heated at 99° C. for 5 minutesand then diluted 10 times. 5 μL of the diluted solution was used as atemplate, the C2hcSalIF and the hIgG1 BamHI L were used, and a cycle of94° C. for 15 seconds, 60° C. for 30 seconds, and 68° C. for 2 minuteswas repeated 25 times. This reaction solution was subjected to 0.8%agarose gel electrophoresis, and the PCR amplification product waspurified by the QIAquick gel extraction kit. This PCR-amplified DNAfragment was digested with SalI and SmaI and the digested product wasintroduced into the N5KG1-Val Lark vector that had been cleaved by thesame enzymes and containing the C2 light chain gene described above. TheDNA nucleotide sequence of the inserted portion was determined and itwas confirmed that the sequence that had been amplified by PCR andinserted was identical to the gene sequence used as a template. Theobtained vector was named N5KG1-Val C2IgμG1. The binding activity of theC2IgμG1 antibody was determined by determining the binding activity ofthe recombinant obtained by gene introduction of the N5KG1-Val C2IgμG1into the FreeStyle293 cells described below to thehCD98/hLAT1-expressing cell line.

The DNA sequences containing the heavy chain variable region and thelight chain variable region of K3, 1-40-1, and 3-69-6 and the amino acidsequences containing the heavy chain variable region and the light chainvariable region were sequences represented by the following sequencenumbers, respectively.

<Nucleotide Sequence of the K3 Heavy Chain Variable Region> (SEQ ID NO:28)

AGAGAGAGAGGTCGACCACCATGGGGTCAACCGCCATCCTCGCCCTCCTCCTGGCTGTTCTCCAAGGAGTCTGTGCCGAGGTGCAGCTGGTGCAGTCTGGAGCAGAAGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGGTTTACCGACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCTTCTATCCTGGTGACTCTGATGCCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAACACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTATTGTGCGAGACGGCGAGATATAGTGGGAGGTACTGACTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA<Amino Acid Sequence of the K3 Heavy Chain Variable Region> (SEQ ID NO:29)

MGSTAILALLLAVLQGVCAEVQLVQSGAEVKKPGESLKISCKGSGYRFTDYWIGWVRQMPGKGLEWMGIFYPGDSDARYSPSFQGQVTISADKSINTAYLQWSSLKASDTAMYYCARRRDIVGGTDYWGQGTLVTVSS<Nucleotide Sequence of the K3 Light Chain Variable Region> (SEQ ID NO:30)

AGAGAGAGAGATCTCTCACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGACTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAGCAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGATCACCTTCGGCCAAGGGA CACGACTGGAGATTAAA<K3 Light Chain Variable Region> (SEQ ID NO: 31)

MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLDWYQQKPGQAPRLLIYDASSRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIK<Nucleotide Sequence of the 1-40-1 Heavy Chain Variable Region> (SEQ IDNO: 32)

AGAGAGAGAGGTCGACCACCATGGAGTTTGGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGGCATGACCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGAGTGGGTCTCTACTATTAGTTGGAATGGTGGTGGCACAGGTTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGGGATATTGTATTATTACCGGCTGCTATGCGGACTACTGGGGCCAGGGAACCCTGGTCA CCGTCTCCTCA<Amino Acid Sequence of the 1-40-1 Heavy Chain Variable Region> (SEQ IDNO: 33)

MEFGLSWVFLVAILKGVQCEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMTWVRQAPGKGLEWVSTISWNGGGTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAGYCIITGCYADYWGQGTLVTVSS<Nucleotide Sequence of the 1-40-1 Light Chain Variable Region> (SEQ IDNO: 34)

AGAGAGAGAGATCTCTCACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTGAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGTGGACGTTCGGCCAAGGGA CCAAGGTGGAAATCAAA<Amino Acid Sequence of the 1-40-1 Light Chain Variable Region> (SEQ IDNO: 35)

MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWWTFGQGTKVEIK<Nucleotide Sequence of the 3-69-6 Heavy Chain Variable Region> (SEQ IDNO: 36)

GTCGACCCACCATGGACTGGACCTGGAGCATCCTTTTCTTGGTGGCAGCAGCAACAGGTGCCCACTCCCAGGTTCAACTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGTAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGATGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAATACGAACTATGTACAGAAGTTCCAGGACAGAGTCACCATGACCAGAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATCGGGGCAGCAATTGGTATGGGTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGT CTCCTCA<3-69-6 Heavy Chain Variable Region> (SEQ ID NO: 37)

RRPTMDWTWSILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWMRQAPGQGLEWMGWISAYNGNTNYVQKFQDRVTMTRDTSTSTAYMELRSLRSDDTAVYYCARDRGSNWYGWFDPWGQGTLVT VSS<Nucleotide Sequence of the 3-69-6 Light Chain Variable Region> (SEQ IDNO: 38)

AGATCTCTCACCATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCGTACACTTTTGGCCAGGGGACCA AGCTGGAGATCAAA<Amino Acid Sequence of the 3-69-6 Light Chain Variable Region> (SEQ IDNO: 39)

RSLTMETPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSYTFGQGTKLEIK

The DNA sequences containing the C2 heavy chain variable region and thelight chain variable region and the amino acid sequences containing theheavy chain variable region and the light chain variable region areshown below, respectively.

5<Nucleotide Sequence of the C2IgG1 Heavy Chain Variable Region> (SEQ IDNO: 40)

GTCGACCACCATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCCCAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGAGTACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAGCCAGTTCTTCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGACAAGGGACGGGGCTCGCCCTATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCT CA<Amino Acid Sequence of the C2IgG1 Heavy Chain Variable Region> (SEQ IDNO: 41)

STTMKHLWFFLLLVAAPRWVLSQLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKSQFFLKLSSVTAADTAVYYCARQGTGLALFDYWGQGTLVTVSS<Nucleotide Sequence of the C2IgG1Ns Heavy Chain Variable Region> (SEQID NO: 42)

GTCGACCACCATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCCCAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGAGTACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGACAAGGGACGGGGCTCGCCCTATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCT CA<Amino Acid Sequence of the C2IgG1Ns Heavy Chain Variable Region> (SEQID NO: 43)

STTMKHLWFFLLLVAAPRWVLSQLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARQGTGLALFDYWGQGTLVTVSS<Nucleotide Sequence from the C2IgμG1 Heavy Chain Variable Region to theBinding Site to the Human IgG1> (SEQ ID NO: 44)

GTCGACCACCATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCCCAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGAGTACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAGCCAGTTCTTCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGACAAGGGACGGGGCTCGCCCTATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTCCTGTGAGAATTCCCCGTCGGATACGAGCAGCGTGGCCGTT<Amino Acid Sequence from the C2IgμG1 Heavy Chain Variable Region to theBinding Site to the Human IgG1> (SEQ ID NO: 45)

STTMKHLWFFLLLVAAPRWVLSQLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKSQFFLKLSSVTAADTAVYYCARQGTGLALFDYWGQGTLVTVSSGSASA PTLFPLVSCENSPSDTSSVAV<Nucleotide Sequence of the Light Chain Variable Region of C2IgG1 andC2IgμG1> (SEQ ID NO: 46)

AGATCTCTCACCATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTTCTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTCGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTATATTCACTTTCGGCCCTGG GACCAAAGTGGATATCAAA<Amino Acid Sequence of the Light Chain Variable Region of C2IgG1 andC2IgμG1> (SEQ ID NO: 47)

RSLTMETPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPIFTFGPGTKVDIK

The light chain variable regions and the heavy chain variable regions ofK3 and C2IgG1 (namely, nucleic acids of the sequences represented by SEQID NOs: 28 and 30 and SEQ ID NOs: 40 and 46) among the above antibodysequences were introduced into the pCR4Blunt-TOPO vector and theresultants were deposited to the International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology, and given the accession numbers of FERM BP-10552 (indicationfor identification: K3/pCR4) and FERM BP-10551 (indication foridentification: C2IgG1 /pCR4).

The respective antibody variable regions contains the heavy chain andthe light chain and also the restriction enzyme recognition sequenceused for binding and isolation. The light chain variable regions of therespective antibodies can be isolated using restriction enzymes BglIIand BsiWI, and the heavy chain variable regions can be isolated usingrestriction enzymes SalI and NheI. The gene sequences of thosecontaining the respective antibody variable regions inserted into thepCR4Blunt-TOPO vector, the restriction-enzyme restriction site, and thelike are shown below.

<K3/pCR4> (SEQ ID NO: 48)

AGAGAGAGAGATCTCTCACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGACTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAGCAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGTACGCTCTCTCTCTAGAGAGAGAGGTCGACCACCATGGGGTCAACCGCCATCCTCGCCCTCCTCCTGGCTGTTCTCCAAGGAGTCTGTGCCGAGGTGCAGCTGGTGCAGTCTGGAGCAGAAGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGGTTTACCGACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCTTCTATCCTGGTGACTCTGATGCCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAACACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTATTGTGCGAGACGGCGAGATATAGTGGGAGGTACTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCT AGCCTCTCTCTCT<C2IgG1 /pCR4> (SEQ ID NO: 49)

AGAGAGAGAGATCTCTCACCATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTTCTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTCGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTATATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGCTCTCTCTCTAGAGAGAGAGGTCGACCACCATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCCCAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGAGTACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAGCCAGTTCTTCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGACAAGGGACGGGGCTCGCCCTATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGT CTCCTCAGCTAGCCTCTCTCTCT

Example 7 Preparation of Recombinant Antibody

The recombinant antibody expression vector constructed in Example 6 wasintroduced into host cells to prepare recombinant antibody-expressingcells. As the host cells for expression, a dhfr-deficient cell line(ATCC CRL-9096) of CHO cells was used. The vector was introduced intothe host cells by electroporation. About 2 μg of the antibody expressionvector was linearized by restriction enzymes, the gene was introducedinto 4×10⁶ CHO cells under the conditions of 350V and 500 μF using aBio-Rad electrophoreter, and the cells were inoculated to a 96-wellculture plate. The agent corresponding to a selection marker of theexpression vector was added and the cells were continuously cultured.After checking appearance of colonies, the antibody-expressing cell linewas screened by the method described in Example 4. The antibody waspurified from the screened cells in accordance with the method inExample 8. In addition, the recombinant antibody expression vector wasintroduced into the FreeStyle293 cells (manufactured by Invitrogen) inaccordance with the attached instruction manual to express a recombinantantibody.

Example 8 Purification of Antibody

A hybridoma culture supernatant containing human IgG antibody wasprepared by the method described below. The antibody-producing hybridomawas acclimated in an eRDF medium (manufactured by Kyokuto PharmaceuticalIndustrial Co., Ltd.) containing bovine insulin (5 μg/mL, manufacturedby Gibco), human transferin (5 μg/mL, manufactured by Gibco),ethanolamine (0.01 mM, manufactured by Sigma), and sodium selenite(2.5×10⁻⁵ mM, manufactured by Sigma). The hybridoma was cultured in atissue culture flask, and the culture supernatant was collected when theviable rate of the hybridoma was 90%. The collected supernatant wasfiltered through 10 μm and 0.2 μm filters (manufactured by GelmanScience) to remove contaminants such as the hybridoma and the like. Theculture supernatant containing the antibody was affinity-purified usingProtein A (manufactured by Amersham), PBS as an absorption buffer, and20 mM sodium citrate buffer (pH 3.0) as an elution buffer. The elutionfractions were adjusted to around pH 6.0 by adding 50 mM sodiumphosphate buffer (pH 7.0). The prepared antibody solution was replacedwith PBS using a dialysis membrane (10,000 cut, manufactured by SpectrumLaboratories) and filter-sterilized through a membrane filter MILLEX-GV(manufactured by Millpore) having a pore size of 0.22 μm to yield thepurified antibody. The concentration of the purified antibody wasobtained by measuring the absorbance at 280 nm and converting a measuredvalue at 1.45 Optimal density to 1 mg/mL.

Example 9 Specificities of Each of the Monoclonal Antibodies

Reactivities of the respective monoclonal antibodies obtained in Example4 were examined by the same method as the FACS analysis clearlydescribed in Example 5. The cell lines prepared in Example 2 were usedto prepare a cell suspension at 2×10⁶/mL using a Staining Buffer (SB)and the cell suspension was dispensed in a 96-well round-bottomed plate(manufactured by Becton Dickinson) at 50 μL/well. The concentration ofeach of the recombinant antibodies prepared in Example 4 to Example 8was adjusted to 5 μg/mL using SB, and 50 μL of the antibody solution wasadded to the respective wells and stirred. An anti-dinitrophenyl (DNP)human IgG1 antibody prepared in KM mice was used as a negative control.After reaction at ice temperature for 30 minutes, the mixture wascentrifuged (2000 rpm, 4° C., 2 minutes) to remove the supernatant. Thepellets were washed once with 100 μL/well of SB, then a 200-time dilutedRPE fluorescently-labeled rabbit anti-human IgK F(ab′)₂ antibody(manufactured by Dako Cytomation) was added at 50 μL/well and theresultant solution was reacted at ice temperature for 30 minutes. Afterwashing with SB once, the resultant pellets were suspended in 300 μL ofSB, the fluorescence intensity showing the binding of the antibody wasmeasured by the FACS.

As a result, all the antibodies exhibited strong binding activity to thehCD98/hLAT1-E-expressing CT26 cells (FIG. 1) or thehCD98/hLAT1-E-expressing L929 cells (FIG. 2), while no binding activityto CT26 cells or L929 cells was observed. Further, any of the antibodiesdid not bind to the hLAT1-E-expressing L929 cells, but they bound to thehCD98-expressing L929 cells. It was found accordingly that the bindingsite of the C2, K3, 7-95-8, 10-60-7, 3-69-6, and 1-40-1 antibodies waslocated at hCD98 (FIG. 2).

Example 10 Regions of hCD98 Protein Involved in Antigen Binding of Eachof the Monoclonal Antibodies

The region of the hCD98 molecule that is important for binding of eachof the monoclonal antibodies was examined.

First, reactivity to a tunicamycin-treated K562 cell line was examined.2×10⁵ K562 cells were inoculated to a 6-well plate (4 mL/well) andcultured at 37° C. in 5% CO₂ for 72 hours in the presence/absence of 5μg/mL tunicamycin (manufactured by Sigma). It was confirmed by Westernblotting that the molecular weight of hCD98, whose original molecularweight was about 80 Kda, was about 60 kDa under this condition, whichcorresponded to a theoretical value after removal of the N-linkedcarbohydrate chain. The cells were collected after culturing andsuspended at 2×10⁶/mL in a Staining Buffer (SB). The cell suspension wasdispensed in a 96-well round-bottomed plate (manufactured by BectonDickinson) at 50 μL/well. Each of the recombinant antibodies prepared at5 μg/mL using SB was added at 50 μL/well and the resultant solution wasreacted at ice temperature for 30 minutes. An anti-DNP human IgG1antibody was used as a negative control. After washing once with SB, anRPE fluorescently-labeled goat anti-human Igy F(ab′)₂ antibody(manufactured by SuthernBiotech) diluted 200 times with SB was added andthe mixture was incubated at ice temperature for 30 minutes. Afterwashing once with SB, the cells were suspended in 300 μL of FACS buffer,the fluorescence intensity showing the binding of the antibody wasmeasured by the FACS.

As a result, no decrease in binding activity to thetunicamycin-untreated K562 cells as compared with the binding activityto the untreated cells was observed for any of the antibodies (FIG. 3).The above results show that the binding site of the respectiveantibodies was not the N-linked carbohydrate chain, strongly suggestingthat these monoclonal antibodies were an hCD98 antibody. Further, theregion of hCD98 that is important for the binding of the respectivemonoclonal antibodies was examined in Example 11.

Example 11 Region of Human CD98 Protein Important for Binding Reactionof Each of the Antibodies

Since each of the antibodies did not have cross-reactivity to mouse CD98(mCD98), a chimera CD98 prepared by artificially binding of mCD98 andhCD98 was utilized to examine a region of human CD98 protein importantfor a binding reaction of each of the antibodies.

The chimera CD98 was prepared as described below. Based on the sequenceinformation about mCD98 and hCD98, EcoRI hCD98U (5′-CCG GAA TTC cCa cCaTGA GCC AGG ACA CCG AGG TGG ATA TGA-3′ (SEQ ID NO: 50)), NotI hCD98(5′-AAG GAA AAA AGC GGC CGC TCA TCA GGC CGC GTA GGG GAA GCG GAG CAGCAG-3′ (SEQ ID NO: 51)), EcoRI mCD98 (5′-CCG GAA TTC CCA CCA TGA GCC AGGACA CCG AAG TGG ACA TGA AA-3′ (SEQ ID NO: 52)), NotI mCD98L (5′-AAG GAAAAA AGC GGC CGC TCA TCA GGC CAC AAA GGG GAA CTG TAA CAG CA-3′ (SEQ IDNO: 53)), cCD98 D2-F (5′-TCA TTC TGG ACC TTA CTC CCA ACT ACC-3′ (SEQ IDNO: 54)), cCD98 D2-R (5′-GGT AGT TGG GAG TAA GGT CCA GAA TGA-3′ (SEQ IDNO: 55)), cCD98 D3-F (5′-TGC TCT TCA CCC TGC CAG GGA CCC CTG TTT T-3′(SEQ ID NO: 56)), and cCD98 D3-R (5′-AAA ACA GGG GTC CCT GGC AGG GTG AAGAGC A-3′ (SEQ ID NO: 57)) were synthesized. In PCR, mCD98(GenBank/EMBL/DDBJ accession no. U25708), a plasmid vectorpcDNA3.1-mCD98 retaining cDNA encoding human CD98, and pEF6/hCD98prepared in Example 1 were used as templates.

KOD-Plus of Toyobo was used for amplification of cDNA. A reactionsolution having a composition of 15 μL of cDNA, 5 μL of 10xKOD-PlusBuffer, 5 μL of dNTP mix, 1 μL of KOD-Plus, 3 μL of 25 mM MgSO4, a Fprimer, and a R primer was prepared in a final volume of 50 μL usingdouble distilled water and subjected to PCR.

cDNA 1, F primer 1, and R primer 1, or cDNA 2, F primer 2, and R primer2 were used, and a cycle of 94° C. for 15 seconds, 60° C. for 30seconds, and 68° C. for 90 seconds (a cycle of 94° C. for 15 seconds,55° C. for 30 seconds, and 68° C. for 50 seconds) was repeated 25 times.This reaction solution was subjected to 0.8% agarose gelelectrophoresis, and the PCR amplification product was purified by aQIAquick gel extraction kit. The PCR amplification products were namedP1 and P2, respectively. Each 5 μL of 2 to 3-time diluted P1 and P2 wasthen placed, and a cycle of 94° C. for 15 seconds, 55° C. for 30seconds, and 68° C. for 2 minutes was repeated 3 times in the absence ofa primer. After this reaction solution was heated to 99° C. for 5minutes, the solution was diluted 5 to 10 times. 5 μL of this solutionwas used as a template together with F primer 1 and R primer 2, and acycle of 94° C. for 15 seconds, 60° C. (55° C.) for 30 seconds, and 68°C. for 2 minutes was repeated 25 times. This reaction solution wassubjected to 0.8% agarose gel electrophoresis, and the PCR amplificationproduct was purified by the QIAquick gel extraction kit.

Chimera CD98-1, chimera CD98-2, and chimera CD98-3 were prepared usingthe following combinations (cDNA 1: F primer 1: R primer 1; and cDNA andF primer 2: R primer 2): (pEF6/hCD98: EcoRIhCD98U: cCD98D2-R; andpcDNA3.1-mCD98: cCD98D2-F: NotImCD98L); (pEF6/hCD98: EcoRIhCD98U:cCD98D3-R; and pcDNA3.1-mCD98: cCD98D3-F: NotImCD98L); and(pcDNA3.1-mCD98: EcoRImCD98U: cCD98D2-R; and pEF6/hCD98: cCD98D2-F:NotIhCD98L). The respective PCR-amplified cDNA fragments were digestedwith EcoRI and NotI and ligated to a pEF6myc-His/Bsd vector(manufactured by Invitrogen) that had been cleaved by the same enzymes.The DNA nucleotide sequence of the inserted portion was determined andit was confirmed that the sequence that had been amplified by PCR andinserted was identical to the gene sequence used as a template. Therespective vectors were expressed in L929 cells together with thepEF1/hLAT1-EGFP vector prepared in Example 1 by the same method as inExample 2, and the binding of the respective FITC-labeled antibodies wasexamined by the FACS analysis by the same method as in Example 10. As aresult (FIG. 4), the K3, 7-95-8, 10-60-7, and 3-69-6 antibodies boundonly to L929 cells expressing the chimera CD98-3, similarly to thecommercially available FITC-labeled anti-human CD98 antibody (cloneUM7F8, manufactured by Becton Dickinson Ca. No. 556076), and it wassuggested that the region from the amino acid residue 372 to the aminoacid residue 530 of hCD98 is important for binding of these antibodies.On the other hand, the C2 antibody and the 1-40-1 neutralized antibodybound strongly only to the chimera CD98-2, showing that the region fromthe amino acid residue 104 to the amino acid residue 371 of hCD98 isimportant for the binding of these antibodies.

Example 12 Amino Acid Uptake Suppression Activity of Each of theMonoclonal Antibodies

In order to determine whether or not the monoclonal antibodiesinfluenced amino acid uptake of human bladder cancer cell line T24cells, a substrate uptake experiment was conducted using leucine as asubstrate in accordance with the method of Kanai et al. (Kim et al.,Biochim. Biophys. Acta 1565: 112-122, 2002) as described below. 1×10⁵cells of the T24 cell line were inoculated to a 24-well culture plateand cultured in an MEM medium (manufactured by SIGMA ALDRICH) containing10% FCS at 37° C. in 5% CO₂ for 2 days. After the culturing, the mediumwas removed, 0.25 mL/well of HBSS(−)(Na+-free) containing 200 μg/mL ofthe antibody was added and the cells were cultured at 37° C. in 5% CO₂for 10 minutes. The recombinant antibodies were used for the C2, K3,7-95-8, 10-60-7, 3-69-6, 1-40-1, and anti-DNP human antibodies and theantibody derived from a hybridoma was used for the 5-80-1. After that,the supernatant was removed, 0.5 mL/well of HBSS(−)(Na+-free) containing1 μM ¹⁴C-Leu (manufactured by MORAVEK BIOCHEMICALS) was added and thecells were cultured for 1 minute. After washing with an ice-cooledHBSS(−)(Na+-free) solution 3 times, 0.1 N sodium hydroxide was added at0.5 mL/well and the cells were collected. The amount of ¹⁴C-Leu in thecollected solution was measured with a liquid scintillation countermodel LSC-5100 (manufactured by ALOKA). The ¹⁴C-Leu uptake of therespective cells was obtained by measuring the protein concentration ofthe collected solution by the BCA method and standardizing the obtainedvalue by the protein amount. The results (FIG. 5) show that the 1-40-1,K3, C2IgG1, 10-60-7, and 3-69-6 significantly suppressed leucine uptakeas compared with the control antibody (DNP human antibody). Thefollowing experiments were conducted using the 1-40-1, K3, C2IgG1,10-60-7, and 3-69-6 that significantly suppressed leucine uptake.

Example 13 Fluorescence Labeling of Each of the Monoclonalanti-hCD98/hLAT1 Antibodies

Each of the antibodies was fluorescently labeled by the method describedbelow. A fluorescent substance, fluorescein isothiocyanate (FITC,manufactured by Sigma), was bound to the respective recombinantantibodies prepared in Example 4 to Example 8 in accordance with theattached instruction manual. To 1 to 2 mg/mL of the antibody in 200 mMsodium carbonate buffer (pH 8.3 to 8.5), FITC dissolved in dimethylformamide was added in an amount of 20 to 40 times that of the antibodymolecule, and the mixture was reacted while stirring at room temperaturefor 2 to 3 hours. The mixture was applied to a gel filtration column(NAP5, manufactured by Amersham Pharmacia Biotech) equilibrated with PBSto remove FITC that did not bind to the antibody. Under this condition,about three FITCs bound to 1 molecule of the antibody. All thefluorescently-labeled antibodies bound to a human colorectal cancerDLD-1 cell line that had been confirmed to express hCD98.

Example 14 Reactivity of Each of the Monoclonal Antibodies to HumanPeripheral Blood-Derived T Cells, B Cells, and Monocytes and NormalHuman Aortic Endothelial Cells (HAEC)

CD98 was known to be expressed in monocytes, activated T cells, andcultured normal endothelial cells. Thus, reactivities of the respectiveantibodies to human peripheral blood-derived T cells, B cells, andmonocytes and human aortic endothelial cells (HAEC) were determined. Thehuman peripheral blood-derived cells were prepared by the followingmethod. 10 mL of human peripheral blood containing 1 mL of heparin(manufactured by Novo) was diluted 2 times with PBS, overlaid on 20 mLof a Ficoll-Paque PLUS solution (manufactured by Amersham PharmaciaBiotech), and centrifuged at 1500 rpm for 30 minutes, and then the cellswere collected. After washing with PBS 2 times, mononulear cells wereprepared. Part of the mononulear cells was cultured in an RPMI medium(manufactured by Gibco) containing 10 μg/mL of phytohaemagglutinin(manufactured by Sigma, PHA), 10% FCS, 0.1 mM non-essential amino acidsolution (manufactured by Gibco), 5.5×10⁻⁶ M 2-mercaptoethanol(manufactured by Gibco), and Penicillin/Streptomycin/Glutamine(manufactured by Gibco) at 37° C. in 5% CO₂ for 72 hours. Expression ofCD25 that was an activation marker was observed for human peripheralblood-derived T cells and B cells by PHA stimulation (FACS analysisusing an FITC-labeled anti-human CD25 antibody (manufactured by BectonDickinson Ca. 555431)). The respective prepared cells were suspended inthe a Staining Buffer (SB) at 2×10⁶/mL, and the cell suspension wasdispensed in a 96-well round-bottomed plate (manufactured by BectonDickinson) at 50 μL/well. The respective FITC-labeled antibodiesprepared in Example 13 at 5 μg/mL was reacted with an anti-human CD3antibody (manufactured by Becton Dickinson Ca. No. 555340), ananti-human CD14 antibody (manufactured by Becton Dickinson Ca. No.347497), or an anti-human CD19 antibody (manufactured by Immunotech Ca.No. IM1285) at ice temperature for 30 minutes. The commerciallyavailable FITC-labeled anti-human CD98 antibody (clone UM7F8) was usedas a positive control, and the FITC-labeled anti-DNP human IgG1 antibodywas used as a negative control. After washing with SB once, theresultant was suspended in 300 μL of FACS buffer and the reactivities ofthe respective antibodies were determined by FACS.

As a result, the antibodies other than C2IgG1 exhibited a binding modesimilar to UM7F8, thus bound significantly to the monocytes, activated Tcells, and activated B cells (FIG. 6 and FIG. 7). On the other hand,C2IgG1 was not observed to bind significantly to any of the cells (FIG.6 and FIG. 7).

HAEC cells (manufactured by Cambrex) were cultured in accordance withthe attached instruction manual and then the cells subcultured not morethan 4 times were used. The reactivities of C2IgG1, K3, 7-95-8, 10-60-7,3-69-6, and 1-40-1 antibodies to the cultured HAEC were examined by thesame method as described above. When the respective antibodies werereacted at the concentrations of 3.2 ng/mL to 50 μg/mL, the K3, 7-95-8,10-60-7, 3-69-6, and 1-40-1 bound to HAEC, but C2IgG1 did not bind toHAEC (FIG. 8).

It was shown, on the other hand, that all the antibodies had higherspecificity to DLD-1 cancer cells than UM7F8 under certain condition (anantibody concentration of 3 μg/mL or lower in the present Example) whenthe antibodies were reacted with the human colorectal cancer cell lineDLD-1 under the same condition (FIG. 8). It was strongly suggested thatC2IgG1, in particular, was an antibody having high cancer specificity.The experiment described below was conducted using the C2IgG1, K3, and3-69-6.

Example 15 Reactivity of Each of the Monoclonal Antibodies to CancerCell Lines

The reactivities of the respective antibodies of C2IgG1, K3, and 3-69-6to the colorectal cancer cell line (DLD-1), a lung cancer cell line(H226), a prostate cancer cell line (DU145), melanoma cell lines (G361,SKMEL28, and CRL1579), a non-Hodgkin lymphoma cell line (Ramos), abladder cancer cell line (T24), breast cancer cell lines (MCF andMDA-MB-231), a pancreatic cancer cell line (HS766T), a multiple myelomacell line (IM9), and an erythroblastic leukemia cell lines (see FIG. 3for K562) were examined by the FACS analysis by the same method as inExample 9. The cell suspension at 2×10⁶/mL was prepared with a StainingBuffer (SB) for the cell lines and dispensed in a 96-well round-bottomedplate (manufactured by Becton Dickinson) at 50 μL/well. The antibody orthe FITC-labeled antibody prepared to 5 μg/mL was added at 50 μL/welland allowed to react at ice temperature for 30 minutes. The anti-DNPhuman IgG1 antibody or the FITC-labeled anti-DNP human IgG1 antibody wasused as a negative control. After washing with SB once, 50 μL of the RPEfluorescently-labeled goat anti-human Igy F(ab′)₂ antibody (manufacturedby SuthernBiotech) diluted 200 times with SB was added and the mixturewas incubated at ice temperature for 30 minutes. In the case of theFITC-labeled antibody, this operation was omitted. After washing with SBonce, the resultant was suspended in 300 μL of FACS buffer and theaverage fluorescence intensity of the respective cells was measured byFACS.

As a result, all the antibodies were found to have binding activity tothe respective cancer cell lines (FIG. 9 and FIG. 10). In addition, allthe antibodies bound strongly to the human colorectal cancer cell lineof Colo205, SW480, SW620, LOVO, LS180, and HT29.

Example 16 Anti-Tumor Effect of K3, C2IgG1, and 3-69-6 in Cancer MouseModel

The anti-tumor effect of the recombinant monoclonal antibodies of K3,C2IgG1, and 3-69-6 prepared in Example 4 to Example 8 were examinedusing a cancer mouse model in accordance with the method describedbelow.

5-week old Balb/c nude mice (purchased from Clea Japan) were allocatedinto groups consisting of 5 mice based on the individual body weight. Amixture of 5×10⁶ colorectal cancer Colo205 cells and 5 μg of theantibody in 100 μL of PBS was subcutaneously transplanted in theabdomen. On days 2, 4, and 6 after transplantation, the antibodydissolved in a solvent (PBS containing 1% mouse serum) at 100 μg/100 μLwas administered intraperitoneally to the mice and a tumor size wasmeasured. The solvent was used as a negative control for the antibody.

The results of the experiment above are shown in FIG. 11. The respectivebroken lines in the FIG. show data for the individual mice. In thecontrol group, engraftment of cells of the cancer cell line was observedin all the individuals on day 5 and the average tumor volume (calculatedby long diameter x short diameter x short diameter×0.5)±SE was165.55±31.71 mm³ on day 12. In the C2IgG1 group, on the other hand, onlyone individual exhibited tumor growth as in the control group (a tumormass of 169.44 mm³ on day 12), but a stronger anti-tumor activity ofadministration of the C2IgG1 antibody was observed in other individuals.The averages volume±SE of the tumor masses on day 28 were 1977.64±442.04for the control group and 775.31±622.47 for the C2IgG1 antibodyadministration group, thus the C2IgG1 antibody suppressed significantlythe growth of the Colo205 cancer cell-derived tumor (p<0.01). Noengraftment of cancer was observed in any individuals of the K3 group orthe 3-69-6 group even after 30 days or more had passed. The average bodyweight decreased only in the control group (a decrease by about 20% ascompared with the K3 group on day 30 after transplantation).

Based on these results, the K3, C2IgG1, and 3-69-6 were found to beantibodies with cancer cell growth suppressing activity.

Example 17 Anti-Tumor Effect of C2IgG1 Monoclonal Antibody inCancer-Bearing Isogenic Mouse Model

The anti-tumor effect of the recombinant monoclonal antibody C2IgG1prepared in Example 4 to Example 8 was examined in a cancer-bearingisogenic mouse model in accordance with the method described below.

Balb/c female mice to which the hCD98/hLAT1-E-expressing CT26 cellsprepared in Example 2 were transplanted at 5×10⁶ cells were divided into2 groups by 5 mice each based on tumor volume. 100 μg/100 μL of theC2IgG1 in a solvent (PBS containing 1% mouse serum) was administeredintraperitoneally to the mice at the point (on day 0) when a tumorvolume increased to about 90 mm3 (calculated by long diameter x shortdiameter x short diameter×0.5), on days 3 and 5. As a control, thesolvent was administered. As a result, C2IgG1 was observed to have anactivity of significantly strongly suppressing the growth of anengrafted tumor (FIG. 12).

Example 18 Cross Reactivities of C2IgG1 and K3 Antibodies to MonkeyCells

Cross-reactivities of the C2IgG1 and K3 to monkey cells (COS-7 cells)were examined by the FACS analysis. 2×10⁶/mL cells were suspended in aStaining Buffer (SB). The cell suspension was dispensed in a 96-wellround-bottomed plate (manufactured by Becton Dickinson) at 50 μL/well.Subsequently, 50 μL of the antibody prepared in 5 μg/mL with SB wasadded, and the resultant was allowed to react at ice temperature for 30minutes. The DNP human IgG1 antibody was used as a negative control.After washing with SB once, 50 μL/well of the RPE fluorescently-labeledgoat anti-human Igy F(ab′)₂ antibody (manufactured by SuthernBiotech)diluted 200 times with SB was added and allowed to react at icetemperature for 30 minutes. After washing with SB once, the resultantwas suspended in 300 μL of FACS buffer and fluorescence intensityshowing antibody binding was measured by FACS. As a result, bothantibodies bound to the COS-7 cell line, and the C2IgG1 and the K3 werefound to be antibodies having cross-reactivity to monkey cells (FIG.13).

Example 19 Effect of C2IgG1 in CANCER-Bearing Mouse Model

The anti-tumor activity of the C2IgG1 was examined using acancer-bearing mouse model in accordance with the method describedbelow.

Burkitt's lymphoma cell line Ramos (purchased from ATCC) wastransplanted subcutaneously at 3×10⁶/mouse individual to the back of6-week old Balb/c-SCID mice (purchased from Clea Japan). On day 13 aftertransplantation, the size of engrafted tumor was measured, andcancer-bearing mice having a tumor of 30 to 140 mm³ were separated intogroups consisting 6 mice/group. The C2IgG1 was administeredintraperitoneally at 100 mg/mouse individual (dissolved in 200 mL ofPBS) 3 times/week. Rituximab (manufactured by Zenyaku Kogyo) was used asa positive control and PBS was used as a negative control. A tumorvolume and body weight were measured 3 times a week. A longer diameter,a shorter diameter, and a height of a tumor mass were measured, and avalue obtained in accordance with the formula of (longerdiameter)×(shorter diameter)×(height)/2 was defined as a tumor volume.

The results are shown in FIG. 14. A significant tumor growth suppressingeffect of the C2IgG1 administration was observed beginning day 16 aftertumor transplantation.

Example 20 Amino Acid-Modified C2IgG1NS

Both of the C2IgG1 and C2IgG1NS have a high aggregate content whenrecombinant antibodies are prepared. Therefore, I (isoleucine) atposition 117 from the fifth M (methionine) as the amino acid at position1 that corresponds to a translation initiation codon ATG in the lightchain variable region sequence of the C2IgG1NS represented by SEQ ID NO:47 was replaced with other amino acids to prepare variants.

Preparation of C2IgG1NS/I117N Vector

In order to prepare C2IgG1NS/I117N in which isoleucine at position 117of the light chain was replaced with asparagine, various mutant DNAsencoding amino acid substitution were prepared using the N5KG1-ValC2IgG1NS vector prepared in Example 6 as a template by the site-specificmutagenesis method with a GeneEditor™ in vitro Site-Directed MutagenesisSystem (Promega No. Q9280).

C2NS Lc 117I/HYND-p: (5′-TCAGTATGGT AGCTCACCTN ATTTCACTTT CGGCCCTGGGACC-3′ (N=A·T·G·C) (SEQ ID NO: 69)) was used as an oligonucleotide(5′-end phosphorylated) for mutagenesis. An intended oligonucleotide formutagenesis and a Selection Oligonucleotide attached to the above kitwere annealed to a template DNA to synthesize mutated chains, and then amutant was selected using the fact that only the mutant grows in thepresence of GeneEditor™ Antibiotic Selection Mix. More specifically, adsDNA template was incubated under an alkaline condition (0.2 M NaOH,0.2 mM EDTA (final concentration)) at room temperature for 5 minutes,then 1/10 volume of 2 M ammonium acetate (pH 4.6) was added forneutralization, and the template was recovered by ethanol precipitation.To the template DNA that had been subjected to alkaline degeneration, anoligonucleotide for mutagenesis, a new Selection Oligonucleotide (BottomSelect Oligo, 5′-end phosphorylated 5′-CCGCGAGACC CACCCTTGGA GGCTCCAGATTTATC-3′ (SEQ ID NO: 85)) for acquisition of antibiotic resistance, andan annealing buffer attached to the kit were added. The mixture was keptat 75° C. for 5 minutes and the temperature was slowly decreased to 37°C. for annealing. Then, for synthesis and ligation of a mutated chain,Synthesis 10× buffer attached to the kit, a T4 DNA Polymerase, and a T4DNA ligase were added and the resultant was allowed to react at 37° C.for 90 minutes. A plasmid DNA was prepared from a transformed E. coliobtained by transforming a competent cells BMH 71-18 mutS in thepresence of the GeneEditor™ Antibiotic Selection Mix and culturing, andthen ElectroMAX DH10B Cells (Invitrogen No. 18290-015) were transformedwith the DNA by the electroporation and inoculated to an LB platecontaining the GeneEditor™ Antibiotic Selection Mix. The transformantgenerated on the plate was cultured, and the plasmid DNA was purifiedand the DNA nucleotide sequence was analyzed. Based on the resultconcerning the DNA nucleotide sequence, an expression vector of C2IgG1NSmutant to which mutation of an intended amino acid was introduced wasobtained. The obtained plasmid DNA expressing the mutant protein withone amino acid substitution was named N5KG1-Val C2IgG1NS/I117N vector.

Preparation of C2IgG1NS/I117C Vector

In order to prepare C2IgG1NS/I117C in which isoleucine at position 117of the light chain was replaced with cystein, various mutant DNAsencoding amino acid substitution were prepared by the site-specificmutagenesis method using GeneEditor™ in vitro Site-Directed MutagenesisSystem (Promega No. Q9280) using the N5KG1-Val C2IgG1NS vector preparedin Example 6 as a template.

C2NS Lc 117I/GRC-p(5′-TCAGTATGGT AGCTCACCTB GTTTCACTTT CGGCCCTGGG ACC-3′(B=C·G·T) (SEQ ID NO: 70)) was used as an oligonucleotide formutagenesis (5′-end phosphorylated). An intended oligonucleotide formutagenesis and a Selection Oligonucleotide attached to the above kitwere annealed with a template DNA to synthesize mutated chain, and thena mutant was selected by using the fact that only the mutant grows inthe presence of GeneEditor™ Antibiotic Selection Mix. More specifically,a dsDNA template was incubated under an alkaline condition (0.2 M NaOH,0.2 mM EDTA (final concentration)) at room temperature for 5 minutes,then 1/10 volume of 2 M ammonium acetate (pH 4.6) was added forneutralization, and the template was recovered by ethanol precipitation.To the template DNA that had been subjected to alkaline degeneration, anoligonucleotide for mutagenesis and a new Selection Oligonucleotide(Bottom Select Oligo, 5′-end phosphorylated 5′-CCGCGAGACC CACCCTTGGAGGCTCCAGAT TTATC-3′ (SEQ ID NO: 85)) for acquisition of antibioticresistance, and an annealing buffer attached to the kit were added, andthen the mixture was kept at 75° C. for 5 minutes and the temperaturewas slowly decreased to 37° C. for annealing. Then, for synthesis andligation of the mutated chain, Synthesis 10× buffer attached to the kit,a T4 DNA Polymerase, and a T4 DNA ligase were added and the resultantwas allowed to react at 37° C. for 90 minutes. A plasmid DNA wasprepared from a transformed E. coli obtained by transforming a competentcells BMH 71-18 mutS in the presence of the GeneEditor™ AntibioticSelection Mix and culturing, and then ElectroMAX DH10B Cells (InvitrogenNo. 18290-015) were transformed with the DNA by the electroporation andinoculated to an LB plate containing the GeneEditor™ AntibioticSelection Mix. The transformant generated on the plate was cultured, andthe plasmid DNA was purified and the DNA nucleotide sequence wasanalyzed. Based on the result concerning the DNA nucleotide sequence, anexpression vector of C2IgG1NS mutant to which mutation of an intendedamino acid was introduced was obtained. The obtained plasmid DNAexpressing the mutant protein with one amino acid substitution was namedN5KG1-Val C2IgG1NS/I117C vector.

Preparation of C2IgG1NS/117IL vector

C2IgG1NS/I117L in which isoleucine at 117 of the light chain wasreplaced with leucine was prepared using the N5KG1-Val C2IgG1NS vectorprepared in Example 6 as a template by the method described below.

For DNA amplification, KOD-Plus of Toyobo was used. A reaction solutionhaving a composition of 1 μL of cDNA, 5 μL of 10xKOD-Plus Buffer, 5 μLof dNTP mix, 1 μL of KOD-Plus, 2 μL of 25 mM MgSO₄, a F primer, and a Rprimer was prepared in a final volume of 50 μL using double distilledwater and subjected to PCR.

C2NS Lc 117IL R (5′-GGTCCCAGGG CCGAAAGTGA ATAGAGGTGA GCTACCATAC TGCTG-3′(SEQ ID NO: 71)) was synthesized, the C2NS Lc 117IL R and the C2-1 LcBgl II F (5′-AGA GAG AGA GAT CTC TCA CCA TGG AAA CCC CAG CGCAGC TTC TCTTC-3′ (SEQ ID NO: 18)) were used, the N5KG1-Val C2IgG1NS vector was usedas a template, and a cycle of 94° C. for 15 seconds, 60° C. for 30seconds, and 68° C. for 1 minute was repeated 25 times. This reactionsolution was subjected to 0.8% agarose gel electrophoresis, and the PCRamplification product was purified by the QIAquick gel extraction kit.This PCR amplification product was named C2NSI117L-F. Next, C2NS Lc117IL F (5′-GCAGTATGGT AGCTCACCTC TATTCACTIT CGGCCCTGGG ACC-3′ (SEQ IDNO: 72)) and C2NS EcoRI R (5′-CCGGAATTCA ACACTCTCCC CTGTTGAAGCTCTTTGTGAC GG-3′ (SEQ ID NO: 73)) were used together with the N5KG1-ValC2IgG1NS vector as a template and a cycle of 94° C. for 15 seconds, 60°C. for 30 seconds, and 68° C. for 1 minute was repeated 25 times. Thisreaction solution was subjected to 0.8% agarose gel electrophoresis andthe PCR amplification product was purified by the QIAquick gelextraction kit. This PCR amplification product was named C2NSI117L-R.Next, 5 μL each of 2-time diluted C2NSI117L-F and C2NSI117L-R wasplaced, and PCR was conduced without primer by repeating a cycle of 94°C. for 15 seconds, 55° C. for 30 seconds, 68° C. 60 seconds 3 times.This reaction solution was heated at 99° C. for 5 minutes and thendiluted 5 times, 5 μL of this solution was used as a template, the C2-1Lc Bgl II F primer and the C2NS EcoRI R primer were used, and a cycle of94° C. for 15 seconds, 55° C. for 30 seconds, and 68° C. 60 seconds wasrepeated 25 times. This reaction solution was subjected to 0.8% agarosegel electrophoresis and the PCR amplification product was purified bythe QIAquick gel extraction kit. This PCR amplified cDNA fragment wasdigested with BglII and EcoRI and introduced into the N5KG1-Val Larkvector that had been cleaved by the same enzymes and contained the aboveC2 heavy chain gene. The DNA nucleotide sequence of the inserted portionwas determined and it was confirmed that the sequence that had beenamplified by PCR and inserted was identical to the gene sequence used asa template. The obtained plasmid DNA expressing the mutant protein withone amino acid substitution mutant was named N5KG1-Val C2IgG1NS/I117Lvector.

Preparation of C2IgG1NS/117IM Vector

C2IgG1NS/I117M in which isoleucine at 117 of the light chain wasreplaced with methionine was prepared using the N5KG1-Val C2IgG1NSvector prepared in Example 6 as a template by the method describedbelow.

For DNA amplification, KOD-Plus of Toyobo was used. A reaction solutionhaving a composition of 1 μL of cDNA, 5 μL of 10xKOD-Plus Buffer, 5 μLof dNTP mix, 1 μL of KOD-Plus, 2 μL of 25 mM MgSO₄, a F primer, and a Rprimer was prepared in a final volume of 50 μL using double distilledwater and subjected to PCR.

C2NS Lc 117IM R (5′-GGTCCCAGGG CCGAAAGTGA ACATAGGTGA GCTACCATAC TGCTG-3′(SEQ ID NO: 74)) was synthesized, the C2NS Lc 117IM R and the C2-1 LcBgl II F (5′-AGA GAG AGA GAT CTC TCA CCA TGG AAA CCC CAG CGCAGC TTC TCTTC-3′ (SEQ ID NO: 18)) were used, the N5KG1-Val C2IgG1NS vector was usedas a template, and a cycle of 94° C. for 15 seconds, 60° C. for 30seconds, and 68° C. for 1 minute was repeated 25 times. This reactionsolution was subjected to 0.8% agarose gel electrophoresis and the PCRamplification product was purified by the QIAquick gel extraction kit.This PCR amplification product was named C2NSI117M-F. Next, C2NS Lc117IM F (5′-GCAGTATGGT AGCTCACCTA TGTTCACTTT CGGCCCTGGG ACC-3′ (SEQ IDNO: 75)) and C2NS EcoRI R (5′-CCGGAATTCA ACACTCTCCC CTGTTGAAGCTCTTTGTGAC GG-3′ (SEQ ID NO: 76)) were used together with the N5KG1-ValC2IgG1NS vector as a template and a cycle of 94° C. for 15 seconds, 60°C. for 30 seconds, and 68° C. for 1 minute was repeated 25 times. Thisreaction solution was subjected to 0.8% agarose gel electrophoresis andthe PCR amplification product was purified by the QIAquick gelextraction kit. This PCR amplification product was named C2NSI117M-R.Then 5 μL each of 2-time diluted C2NSI117M-F and C2NSI117M-R was placedand a cycle of 94° C. for 15 seconds, 55° C. for 30 seconds, and 68° C.for 60 seconds was repeated 3 times in the absence of a primer. Thisreaction solution was heated at 99° C. for 5 minutes and then diluted 5times, 5 μL of this solution was used as a template together with theC2-1 Lc Bgl II F primer and the C2NS EcoRI R primer, and a cycle of 94°C. for 15 seconds, 55° C. for 30 seconds, and 68° C. 60 seconds wasrepeated 25 times. This reaction solution was subjected to 0.8% agarosegel electrophoresis and the PCR amplification product was purified bythe QIAquick gel extraction kit. This PCR-amplified cDNA fragment wasdigested with BglII and EcoRI and introduced into the N5KG1-Val Larkvector that had been cleaved by the same enzymes and contained the aboveC2 heavy chain gene. The DNA nucleotide sequence of the inserted portionwas determined and it was confirmed that the sequence that had beenamplified by PCR and inserted was identical to the gene sequence used asa template. The obtained plasmid DNA expressing the mutant protein withone amino acid substitution was named N5KG1-Val C2IgG1NS/117M vector.

Preparation of Amino Acid-Modified C2IgG1NS

By the method described in Example 7, the C2IgG1NS/I117L vector, theC2IgG1NS/I117M vector, the C2IgG1NS/I117N vector, and the C2IgG1NS/I117Cvector were introduced into FreeStyle293 cells (manufactured byInvitrogen) in accordance with the attached instruction manual toexpress recombinant antibodies. The antibody was purified by the methoddescribed in Example 8 of which part was modified. On day 6, the culturesupernatant was collected and filtered through Steriflip-GP (MILLIPORE,SCGPOO525) to remove contaminants such as cells and the like. Theculture supernatant containing an antibody was affinity purified usingProtein A (manufactured by Amersham), PBS as an absorption buffer, and20 mM sodium citrate buffer (pH 3.4) as an elution buffer. The elutionfractions were adjusted to about pH 5.5 by adding 200 mM sodiumphosphate buffer (pH 7.0). The prepared antibody solution wasconcentrated at 3000 rpm using vivaspin 6 (10 KMW cut VIVA SCIENCE,VS0601), PBS was further added, and the mixture was centrifuged toobtain purified antibody replaced with PBS. The concentration of thepurified antibody was obtained by measuring the absorbance at 280 nm andcalculating 1.45 Optimal density as 1 mg/mL.

Measurement of Content of Aggregate of Amino Acid-Modified C2IgG1 NS

The contents of aggregate of the respective purified antibodies weremeasured using 10 μg (0.1 mg/mL) of the amino acid-modified antibodies.

The content of aggregate of the antibody solution was analyzed by usinga high performance liquid chromatograph (manufactured by Shimadzu),TSK-G3000 SW column (manufactured by Toso), and 20 mM sodium phosphateand 500 mM NaCl pH 7.0 as solvents. Elution positions were compared witha molecular marker for gel filtration HPLC (manufactured by OrientalYeast) (Cat No. 40403701) to identify a monomer and aggregates of theantibody protein, and the content of the aggregate was calculated fromthe respective peak areas.

The results are shown in FIG. 15. FIG. 15 shows that the content ofaggregate was decreased by the amino acid modifications above.

Measurement of Amount of Aggregate of Amino Acid-Modified C2IgG1NS

Reactivities of the amino acid-modified C2IgG1NS antibodies to a tumorcell line, a human CD98/human LAT1 enforced expression cell line, andHAEC by FACS in accordance with the methods described in Examples 14 and15.

The results are shown in FIG. 16A and FIG. 16B. The above aminoacid-modified antibodies, especially C2IgG1NS/I117L, bound to L929 cellsforcibly expressing human CD98 and human LAT1, but did not bind tountreated L929 (FIG. 16A). In addition, these amino acid-modifiedantibodies did not bind to HAEC, but bound to various cancer cells suchas colo204, Ramos, and DLD-1 (FIG. 16B).

Since these results are similar to the binding property of C2IgG1 shownin FIG. 2A and FIG. 8, it is considered that the above aminoacid-modified antibodies, especially C2IgG1NS/I117L, has a low aggregatecontent, has binding specificity to cancer cells similarly to C2IgG1,and may be expected to exhibit anti-tumor activity similarly to C2IgG1.

<Amino Acid Sequence of the Heavy Chain Variable Region of AminoAcid-Modified C2IgG1Ns (Identical to Amino Acid Sequence of C2IgG1 HeavyChain Variable Region)> (SEQ ID NO: 43)

STTMKHLWFFLLLVAAPRWVLSQLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKSQFFLKLSSVTAADTAVYYCARQGTGLALFDYWGQGTLVTVSS<Nucleotide Sequence of the Heavy Chain Variable Region of AminoAcid-Modified C2IgG1Ns (Identical to Nucleotide Sequence of C2IgG1NsHeavy Chain Variable Region)> (SEQ ID NO: 42)

GTCGACCACCATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCCCAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGAGTACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGACAAGGGACGGGGCTCGCCCTATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCT CA<Amino Acid Sequence of the Light Chain Variable Region ofC2IgG1NS/117IL> (SEQ ID NO: 77)

RSLTMETPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLFTFGPGTKVDIK<Nucleotide Sequence of the Light Chain Variable Region ofC2IgG1NS/117IL> (SEQ ID NO: 78)

AGATCTCTCACCATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTTCTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTCGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTCTATTCACTTTCGGCCCTGG GACCAAAGTGGATATCAAA<Amino Acid Sequence of the Light Chain Variable Region ofC2IgG1NS/117IM> (SEQ ID NO: 79)

RSLTMETPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPMFTFGPGTKVDIK<Nucleotide Sequence of the Light Chain Variable Region ofC2IgG1NS/117IM> (SEQ ID NO: 80)

AGATCTCTCACCATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTTCTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTCGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTATGTTCACTTTCGGCCCTGG GACCAAAGTGGATATCAAA<Amino Acid Sequence of the Light Chain Variable Region ofC2IgG1NS/117IN> (SEQ ID NO: 81)

RSLTMETPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPNFTFGPGTKVDIK<Nucleotide Sequence of the Light Chain Variable Region ofC2IgG1NS/117IN> (SEQ ID NO: 82)

AGATCTCTCACCATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTTCTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTCGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTAATTTCACTTTCGGCCCTGG GACCAAAGTGGATATCAAA<Amino Acid Sequence of the Light Chain Variable Region ofC2IgG1NS/117IC> (SEQ ID NO: 83)

RSLTMETPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPCFTFGPGTKVDIK<Nucleotide Sequence of the Light Chain Variable Region ofC2IgG1NS/117IC> (SEQ ID NO: 84)

AGATCTCTCACCATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTTCTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTCGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTTGTTTCACTTTCGGCCCTGG GACCAAAGTGGATATCAAA

1. A human monoclonal antibody or a functional fragment thereof thatbinds CD98 and that has any, pair of sequences of the following (b) to(g) as a heavy chain variable region and a light chain variable region:(b) SEQ ID NOs: 41 and 47, (c) SEQ ID NOs: 43 and 47, (d) SEQ ID NOs: 43and 77, (e) SEQ ID NOs: 43 and 79, (f) SEQ ID NOs: 43 and 81, and (g)SEQ ID NOs: 43 and 83, wherein said functional fragment is selected fromthe group consisting of Fab, Fab′, (Fab′)2, Fv, scFv, and sdFv of saidantibody and a combination thereof.
 2. The human monoclonal antibody ora functional fragment thereof according to claim 1, wherein a subclassof the antibody heavy chain constant region is IgG.
 3. A conjugate,comprising: (i) the human monoclonal antibody or a functional fragmentthereof according to any one of claim 1 or claim 2; and (ii) aheterogeneous domain containing a binding protein, an enzyme, a drug, atoxin, an immunomodulator, a detectable portion or a tag.
 4. A cellexpressing the human monoclonal antibody or a functional fragmentthereof according to any one of claims 1 or
 2. 5. A pharmaceuticalcomposition comprising the human monoclonal antibody or a functionalfragment thereof according to any one of claims 1 or 2 as an activeingredient.
 6. A therapeutic agent for colorectal cancer or colon cancercomprising the human monoclonal antibody or a functional fragmentthereof according to any one of claims 1 or 2 as an active ingredient.7. A method for producing an antibody that binds CD98, comprising:introducing to a host cell an expression vector containing any pair ofsequences of the following (b) to (g): (b) SEQ ID NOs: 40 and 46, (c)SEQ ID NOs: 42 and 46, (d) SEQ ID NOs: 42 and 78, (e) SEQ ID NOs: 42 and80, (f) SEQ ID NOs: 42 and 82, and (g) SEQ ID NOs: 42 and 84, or adegenerate nucleotide sequence thereof; then culturing the host cell toproduce a culture; and obtaining the antibody from the culture.
 8. Themethod according to claim 7, wherein the host cell is selected from thegroup consisting of E. coli, yeast cells, insect cells, mammal cells,and plant cells.